Method of treating or preventing benign prostatic hyperplasia using modified pore-forming proteins

ABSTRACT

The present invention provides a method of treating BPH using modified pore-forming proteins (MPPs). These MPPs are derived from naturally occurring cytotoxic proteins (nPPs) that kill cells by forming pores or channels in the cell membrane, resulting in cell death. The MPPs are generated by modification of the nPPs such that they are capable of being selectively activated at normal prostate cells. Such modification may include the addition of a prostate-specific protease cleavage site to the activation sequence, and/or the addition of a prostate-specific targeting domain to allow selective targeting of prostate cells. These MPPs are capable of selectively targeting and killing normal prostate cells in vivo. The MPPs may be used either alone or in combination with other therapies for the treatment of BPH.

FIELD OF THE INVENTION

The present invention relates to the field of benign prostatichypertrophy, and in particular to the use of modified pore-formingproteins for the treatment of benign prostatic hyperplasia (BPH).

BACKGROUND OF THE INVENTION

Many cytolytic proteins have been described (Lesieur et al. Mol. Membr.Biol. 14:45064, 1997). These naturally occurring cytotoxic proteinsinclude mammalian proteins such as perforin, and bacterial proteins suchas aerolysin (produced by Aeromonas hydrophila), α-hemolysin (producedby Staphylococcus aureus), alpha toxin (produced by Clostridiumsepticum), δ-toxin (produced by Bacillus thuringiensis), anthraxprotective antigen, Vibrio cholerae VCC toxin, Staphylococcusleucocidins, LSL toxin from Laetiporus sulphureus, epsilon toxin fromClostridium perfringens, and hydralysins produced by Cnidaria spp.

Some of these cytotoxic proteins, for example, proaerolysin and alphatoxin, are synthesized as inactive protoxins. These protoxins containdiscrete functionalities including a binding domain, which allowsbinding of the protoxin to a cell, a toxin domain, and either anN-terminal or a C-terminal inhibitory peptide domain that contains aprotease cleavage site. Cleavage of the inhibitory peptide domain at theprotease cleavage site results in activation of the protoxin, leading tooligomerization of the cytotoxin in the plasma membrane, producing poresthat lead to rapid cytolytic cell death (Rossjohn et al. J. Struct.Biol. 121:92-100, 1998). Pore formation physically disrupts the cellmembranes, and results in death of cells in all phases of the cellcycle, including non-proliferating cells (i.e. G₀ arrested). Thesecytotoxins are not specific in the type of cells they are able to kill,as their binding domains target molecules that are found on most cells,and they are generally activated by proteases that are notcell-specific.

Cytolytic pore-forming proteins or modified versions of these proteinshave been proposed as potential therapeutics for the treatment ofcancer. For example, U.S. Pat. No. 5,777,078 describes pore-formingagents that are activated at the surface of a cell by a number ofconditions, including proteolysis, to lyse the cell. These pore-formingagents can be used generally to destroy unwanted cells associated with apathological condition in an animal. Such cells include but are notlimited to tumor cells, cells which are chronically infected with virus,or cells, which when improperly regulated or expressed, result in adisease state, e.g., cells of the immune system. WO 98/020135 describesmethods and compositions relating to Pseudomonas exotoxin proproteinsmodified for selective toxicity. The exotoxin is modified to beactivated by a desired protease by insertion of a protease susceptiblesequence in the proprotein. In one example the exotoxin is modified toinsert a prostate specific antigen (PSA) cleavage site for the purposeof targeting and killing prostate cancer cells.

U.S. Patent Application No. 2004/0235095 describes the use of modifiedcytolytic pore-forming proteins for the treatment of prostate and othercancers. The cytolytic proteins can be modified to include aprostate-specific cleavage site, and/or a prostate-specific targetingdomain and can be used to selectively target and kill prostate cancercells.

Cancer is characterized by an increase in the number of abnormal, orneoplastic cells derived from a normal tissue which proliferate to forma tumor mass, the invasion of adjacent tissues by these neoplastic tumorcells, and the generation of malignant cells which eventually spread viathe blood or lymphatic system to regional lymph nodes and to distantsites via a process called metastasis. In a cancerous state, a cellproliferates under conditions in which normal cells would not grow.Unlike normal cells, in general, cancer cells continue to reproduce,they do not specialize or become mature, and they have the ability tospread from the tissue of origin to other locations within the body.These characteristics of cancer cells generally result from changes inthe relative pattern of gene expression within these cells compared tothat in normal cells. Many strategies for developing therapeutics forthe treatment of cancer have focused on taking advantage of thedifferences in gene expression between normal cells and cancer cells,and targeting cancer cells using molecular markers that are specific tocancer cells.

In contrast, benign prostatic hyperplasia (BPH, also known as benignprostatic hypertrophy) is a non-cancerous condition resulting fromenlargement of the prostate gland as a consequence of the naturalprogression of prostate growth with age. Enlargement of the prostate canbe a result of increased prostate cell proliferation, or an increase inprostate cell size. This progressive prostate growth does not usuallycause problems until late in life. The National Institute of Health(NIH) estimates that 60% of American men in their sixties have somesymptoms of BPH and that the condition affects more than 90% of men intheir seventies and eighties. Approximately 115 million males worldwidein the 50+ age group have varying degrees of BPH. Due to the aging ofthe population, the prevalence is expected to increase substantiallyover the next 20 years. Severe BPH can cause serious problems such asurinary tract infections, bladder and kidney damage, including bladderstones, incontinence and most seriously, gross hematuria and renalfailure due to obstructive uropathy.

There are several strategies currently available for treating BPH. Theseinclude watchful waiting, medical therapy such as alpha blocker therapyand finasteride therapy, balloon dilation and various surgicalprocedures such as transurethral incision of the prostate (TUIP),transurethral resection of the prostate (TURP), and open prostatectomy.Few treatments are without any adverse consequences, and this isparticularly so with treatments for BPH, where there is a delicatebalancing act between the benefits and demerits of the treatmentsavailable. The adverse events following currently available treatmentsfor BPH include impotence (for various surgical procedures ranging fromabout 4% to 40%, the incidence of impotence is also increased after somemedical treatments), incontinence (stress incontinence about 3% aftersurgery, with total urinary incontinence approaching 1%), and the needfor re-treatment. Combined analysis of published data estimated that themean probability for perioperative mortality (death within 90 days of aprocedure) was 1.5% for TURP. For open surgery it was 2.4% and forballoon dilation it was 3.5%.

Currently, the most commonly used hormone therapy is oral administrationof finasteride. Finasteride, commercially available under the tradenameProscar™ from Merck & Co. Inc., Whitehouse Station, N.J., is a synthetic4-azasteroid compound, a specific inhibitor of steroid Type II5α-reductase, and an intracellular enzyme that converts the androgentestosterone into 5α-dihydrotestosterone (DHT). Finasteride helps toshrink the enlarged prostate and reduces elevated PSA due to benignprostate conditions. However, finasteride is known to cause undesirableside effects, which include impotence or lessened desire for sex,problems with ejaculation, and breast enlargement and/or tenderness.Dutasteride (Duagen) is another drug for the treatment of BPH and it iscapable of blocking both types I and II 5α-reductase. Sexual sideeffects are similar to those of finasteride.

Alpha-1 adrenoceptor blocking agents are also currently used forclinical treatment of benign prostatic hyperplasia. Examples includetamsulasin hydrochloride, terazosin hydrochloride, alfuzosinhydrochloride and doxazosin mesylate. The reduction in symptoms of BPHand improvement in urine flow rates following administration of analpha-1 adrenoceptor blocking agent are related to relaxation of smoothmuscle produced by blockage of alpha-1 adrenoceptors in the bladder neckand prostate.

Furthermore, plant sterols and extracts have also been used for thetreatment of benign prostatic hyperplasia.

United States Patent Application No. 20040081659 describes conjugatesuseful to treat BPH comprising 1) oligopeptides with amino acidsequences which are selectively and proteolytically cleaved by PSA,chemically linked to 2) vinca alkaloid cytotoxic agents. Theoretically,the cytotoxic activity of the alkaloid is low in the conjugate andincreased when the linkage is cleaved by PSA.

European Patent Application 0652014 describes a treatment for BPHcomprising administration of PSA (prostate-specific antigen) linked toan immunogenic carrier to induce the production of anti-PSA antibodies.Anti-PSA antibodies may also be used. The immunogenic carrier can betetanus toxin, diphtheria toxin or cholera toxin chain B.

U.S. Pat. No. 6,379,669 describes a method of targeting a specific organby coupling a therapeutic agent to an antibody or fragments thereof.Such coupled therapeutic agents (or immunocongugates) can be used totreat prostate cancer, BPH, or prostatitis. The immunoconjugatesincluded are antibodies against PSA that are linked to various bioactiveagents. The bioactive agents may include bacterial toxins. Similarly, inUnited States Patent Application No. 20020001588, the chemical linkageof antibodies and various bioactive therapeutic agents is exploredfurther.

This background information is provided for the purpose of making knowninformation believed by the applicant to be of possible relevance to thepresent invention. No admission is necessarily intended, nor should beconstrued, that any of the preceding information constitutes prior artagainst the present invention.

SUMMARY OF THE INVENTION

An object of the present invention is to provide a method of treating orpreventing benign prostatic hyperplasia using modified pore-formingproteins.

In accordance with one aspect of the present invention, there isprovided a modified pore-forming protein for use in decreasing prostatesize in a subject, said modified pore-forming protein derived from anaturally-occurring pore-forming protein and comprising one or moreprostate-selective modifications selected from an activation sequencecleavable by a prostate-specific protease, and one or moreprostate-specific targeting domains capable of selectively targetingprostate cells, wherein said modified pore-forming protein is capable ofselectively killing prostate cells.

In accordance with another aspect of the present invention, there isprovided a modified pore-forming protein for use in the treatment ofbenign prostatic hyperplasia (BPH), said modified pore-forming proteinderived from a naturally-occurring pore-forming protein and comprisingone or more prostate-selective modifications selected from an activationsequence cleavable by a prostate-specific protease, and one or moreprostate-specific targeting domains capable of selectively targetingprostate cells, wherein said modified pore-forming protein is capable ofselectively killing prostate cells.

In accordance with another aspect of the present invention, there isprovided a use of a modified pore-forming protein in the preparation ofa medicament for decreasing prostate size in a subject, said modifiedpore-forming protein derived from a naturally-occurring pore-formingprotein and comprising one or more prostate-selective modificationsselected from an activation sequence cleavable by a prostate-specificprotease, and one or more prostate-specific targeting domains capable ofselectively targeting prostate cells, wherein said modified pore-formingprotein is capable of selectively killing prostate cells.

In accordance with another aspect of the present invention, there isprovided a use of a modified pore-forming protein in the preparation ofa medicament for the treatment of benign prostatic hyperplasia (BPH),said modified pore-forming protein derived from a naturally-occurringpore-forming protein and comprising one or more prostate-selectivemodifications selected from an activation sequence cleavable by aprostate-specific protease, and one or more prostate-specific targetingdomains capable of selectively targeting prostate cells, wherein saidmodified pore-forming protein is capable of selectively killing prostatecells.

In accordance with another aspect of the present invention, there isprovided a method of decreasing prostate size in a subject comprisingadministering to said subject an effective amount of a modifiedpore-forming protein, said modified pore-forming protein derived from anaturally-occurring pore-forming protein and comprising one or moreprostate-selective modifications selected from an activation sequencecleavable by a prostate-specific protease, and one or moreprostate-specific targeting domains capable of selectively targetingprostate cells, wherein said modified pore-forming protein is capable ofselectively killing prostate cells.

In accordance with another aspect of the present invention, there isprovided a method of treating benign prostatic hyperplasia (BPH) in asubject, comprising administering to said subject an effective amount ofa modified pore-forming protein, said modified pore-forming proteinderived from a naturally-occurring pore-forming protein and comprisingone or more prostate-selective modifications selected from an activationsequence cleavable by a prostate-specific protease, and one or moreprostate-specific targeting domains capable of selectively targetingprostate cells, wherein said modified pore-forming protein is capable ofselectively killing prostate cells.

In accordance with another aspect of the present invention, there isprovided a modified proaerolysin protein comprising one or moremutations in a large lobe binding domain, and one or moreprostate-specific modifications selected from a prostate-specifictargeting domain capable of selectively targeting prostate cells and anactivation sequence cleavable by a prostate-specific protease, whereinsaid modified proaerolysin is capable of selectively killing prostatecells.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other features of the invention will become more apparent inthe following detailed description in which reference is made to theappended drawings.

FIG. 1 presents a schematic of proaerolysin domains (not drawn to scale)and shows the result of activation by furin.

FIG. 2 depicts a bar graph showing the results of a hemolysis assay inwhich MPP1 is preincubated with human plasma or human plasma spiked withenzymatically active PSA (10,000 ng/ml).

FIG. 3 depicts a graph comparing the in vitro toxicity of several MPPsaccording to embodiments of the invention to that of proaerolysin. TheMPPs are derived from proaerolysin, and include a PSA cleavage site inplace of the native furin site.

FIGS. 4A-4E are schematic drawings (not to scale) showing how aproaerolysin protein can be altered to generate several different MPPsderived from proaerolysin according to embodiments of the presentinvention. The “*” symbol represents one or more point mutations, and/orone or more deletions which decrease proaerolysin binding domainfunction (i.e. the ability to concentrate in a cell membrane).

FIG. 4A represents a schematic drawing of a wild-type proaerolysin. FIG.4B represents a schematic drawing of an MPP derived from proaerolysin,with an activation sequence modified to include a prostate-specificprotease cleavage site. FIG. 4C represents a schematic drawing of an MPPderived from proaerolysin, with an activation sequence modified toinclude one or more prostate-specific protease cleavage sites. FIG. 4Drepresents a schematic drawing of an MPP derived from proaerolysin, withan activation sequence modified to include a prostate-specific proteasecleavage site and with a functionally deleted native binding domain. Thefunctionally deleted native binding domain is generated by one or morepoint mutations or one or more deletions. FIG. 4E represents a schematicdrawing of an MPP derived from proaerolysin, with an activation sequencemodified to include a prostate-specific protease cleavage site and witha functionally replaced native binding domain. The functionally deletednative binding domain is generated as described for FIG. 4D. One or moreprostate-specific targeting domains may be attached at the N-terminus ofthe MPP, or at the C-terminal end of the toxin domain of the MPP, inthis embodiment.

FIG. 5A represents a schematic drawing of an MPP derived fromproaerolysin, with an activation sequence modified to include aprostate-specific protease cleavage site and with a functionallyreplaced native binding domain. The native binding domain is modified byone or more point mutations or one or more deletions. One or moreprostate-specific targeting domains can be optionally attached to theMPP at Y215C, or A300C. FIG. 5B represents a schematic drawing of an MPPderived from proaerolysin, with an activation sequence modified toinclude a prostate-specific protease cleavage site and with afunctionally deleted native binding domain. The native binding domain isfunctionally deleted by deletion of one of the native binding domains ofproaerolysin. FIG. 5C represents a schematic drawing of an MPP derivedfrom proaerolysin, with an activation sequence modified to include aprostate-specific protease cleavage site and with a functionallyreplaced native binding domain. One or more prostate-specific targetingdomains may be attached to either the N-terminus of the toxin domain ofthe MPP, or to the C-terminal end of the toxin domain of the MPP in thisembodiment. One of the native binding domains of the MPP is deleted asdescribed in FIG. 5B. FIG. 5D represents a schematic drawing of an MPPderived from proaerolysin, with an activation sequence modified toinclude a prostate-specific protease cleavage site and with afunctionally replaced native binding domain. One or moreprostate-specific targeting domains may be attached to the MPP at Y215C,or A300C. One of the native binding domains of the MPP is deleted asdescribed in FIG. 5B.

FIG. 6A represents a schematic drawing of an MPP according to oneembodiment of the invention derived from proaerolysin with afunctionally replaced native binding domain. The MPP further comprisesone or more prostate-specific targeting domains. A native binding domainof the MPPP is functionally deleted by mutation or deletion of one ormore amino acid residues. FIG. 6B represents a schematic drawing of anMPP according to one embodiment of the invention derived fromproaerolysin with a functionally replaced native binding domain. The MPPfurther comprises one or more prostate-specific targeting domainsattached to proaerolysin at Y215C or A300C. A native binding domain ofthe MPPP is functionally deleted by mutation or deletion of one or moreamino acid residues. FIG. 6C represents a schematic drawing of an MPPaccording to one embodiment of the invention derived from proaerolysinwith a functionally replaced native binding domain. The MPP furthercomprises one or more prostate-specific targeting domains. The nativebinding domain is functionally deleted by deletion of one of the nativebinding domains of proaerolysin. FIG. 6D represents a schematic drawingof an MPP according to another embodiment of the invention derived fromproaerolysin with a functionally replaced native binding domain. The MPPfurther comprises one or more prostate-specific targeting domainsattached to proaerolysin at Y215C or A300C. The native binding domain isfunctionally deleted by deletion of one of the native binding domains ofproaerolysin.

FIG. 7 depicts a wild-type proaerolysin cDNA sequence (SEQ ID NO:1).

FIG. 8 depicts a wild-type proaerolysin amino acid sequence (SEQ IDNO:2).

FIG. 9 depicts the cDNA sequence (SEQ ID NO:3) of an MPP according toone embodiment of the invention (MPP1), wherein the furin site ofproaerolysin has been replaced with a PSA cleavage site.

FIG. 10 depicts the amino acid sequence (SEQ ID NO:4) of an MPPaccording to one embodiment of the invention (MPP1), wherein the furinsite of proaerolysin has been replaced with a PSA cleavage site.

FIG. 11 depicts the amino acid sequence (SEQ ID NO:5) of a PSA cleavagesite found in human semenogelin I and II proteins.

FIG. 12 depicts the cDNA sequence (SEQ ID NO:6) of an MPP according toone embodiment of the invention (MPP2), wherein the furin site ofproaerolysin has been replaced with a PSA cleavage site.

FIG. 13 depicts the amino acid sequence (SEQ ID NO:7) of an MPPaccording to one embodiment of the invention (MPP2), wherein the furinsite of proaerolysin has been replaced with a PSA cleavage site.

FIG. 14 depicts an example of a PSA cleavage site (SEQ ID NO:8).

FIG. 15 depicts the cDNA sequence (SEQ ID NO:9) of an MPP according toone embodiment of the invention (MPP3), wherein the furin site ofproaerolysin has been replaced with a PSA cleavage site.

FIG. 16 depicts the amino acid sequence (SEQ ID NO:10) of an MPPaccording to one embodiment of the invention (MPP3), wherein the furinsite of proaerolysin has been replaced with a PSA cleavage site.

FIG. 17 depicts a second example of a PSA cleavage site (SEQ ID NO:11).

FIG. 18 depicts the cDNA sequence (SEQ ID NO:12) of an MPP according toone embodiment of the invention (MPP4), wherein the furin site ofproaerolysin has been replaced with a PSA cleavage site.

FIG. 19 depicts the amino acid sequence (SEQ ID NO:13) of an MPPaccording to one embodiment of the invention (MPP4), wherein the furinsite of proaerolysin has been replaced with a PSA cleavage site.

FIGS. 20-27 depict the amino acid sequences of alternative PSA cleavagesites according to the present invention (SEQ ID NOs:14-21,respectively).

FIG. 28 depicts a native luteinizing hormone releasing hormone (LHRH)amino acid sequence (SEQ ID NO:22).

FIG. 29 depicts a modified luteinizing hormone releasing hormone (LHRH)amino acid sequence (SEQ ID NO:23).

FIG. 30 depicts the amino acid sequence (SEQ ID NO:24) of an MPPaccording to one embodiment of the present invention (MPP6), in whichthe furin site of proaerolysin has been replaced with a PSA cleavagesite, and wherein the native binding domain of proaerolysin has beenmodified.

FIG. 31 depicts the amino acid sequence of an MPP according to oneembodiment of the present invention (MPP7), in which the furin site ofproaerolysin is retained, and the native binding domain of proaerolysinhas been deleted and replaced with SEQ ID NO:23 (SEQ ID NO:25).

FIG. 32 depicts the effects of an MPP according to one embodiment of theinvention (MPP5) in the prostate gland of monkeys after treatment for 3days. A and B depict the prostate glands of control monkeys treated withvehicle alone; C and D depict the prostate glands of monkeys treatedwith 1 μg of the MPP; E and F depict the prostate glands of monkeystreated with 5 μg of the MPP; and G and H depict the prostate glands ofmonkeys treated with 25 μg of the MPP.

FIG. 33 depicts the effects of an MPP according to one embodiment of theinvention (MPP5) in the prostate gland of monkeys treated for 15 days. Aand B depict the prostate glands of control monkeys treated with vehiclealone; C and D depict the prostate glands of monkeys treated with 1 μgof the MPP; E and F depict the prostate glands of monkeys treated with 5μg of the MPP; and G and H depict the prostate glands of monkeys treatedwith 25 μg of the MPP.

FIG. 34 depicts the nucleotide sequence of MPP5 (SEQ ID NO:30). The ATGstart codon and TAA stop codon are underlined and in bold. The HindIIIand EcoRI restriction sites are in bold text. The PSA cut site isunderlined and the 6 His tag is in bold italicized text.

FIG. 35 depicts the amino acid sequence of MPP5 (SEQ ID NO:31). Theamino acid sequence was derived from the nucleic acid sequence shown inFIG. 34. Amino acids 427-432 (the PSA cut site) are underlined and inbold. The 6 His tag is in bold text.

FIG. 36 depicts activation of MPP5 in prostate tissue fragmentconditioned media assayed by degree of hydrolysis of washed red bloodcells.

FIG. 37 depicts the ability of sera from various species to cleave MPP5.

FIG. 38 depicts the effect of MPP5 on monkey prostates.

FIG. 39 depicts the humoral response to administration of MPP5 inmonkeys.

FIG. 40 depicts the nucleotide sequence (SEQ ID NO:73) of a wild-typeClostridium septicum alpha toxin.

FIG. 41 depicts the amino acid sequence (SEQ ID NO:74) of a wild-typeClostridium septicum alpha toxin.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the use of modified pore-formingproteins for the treatment of BPH. The MPPs are derived fromnaturally-occurring pore-forming proteins (nPPs) that kill cells byinserting into the membrane and forming pores or channels in the cellmembranes of target cells, resulting in cell death. In one embodiment,the MPP inserts into the cell membrane, irreversibly, and thus bystandercells are not affected. The MPPs comprise prostate-selectivemodifications that result in the ability of the MPPs to selectivelytarget normal prostate cells relative to cells from other tissues. TheMPPs are capable of selectively killing normal prostate cells in vivo,and are capable of decreasing the weight or volume of normal prostategland in vivo. Thus, the MPPs according to the present invention may beused alone, or in combination with other therapies for the treatment ofBPH. This is in contrast to the molecules described in U.S. PatentApplication No. 20040235095 which describes the use of modifiedcytolytic proteins to treat localized or metastatic prostate cancer.

DEFINITIONS

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs.

The techniques and procedures are generally performed according toconventional methods in the art and various general references (seegenerally, Sambrook et al. Molecular Cloning: A Laboratory Manual, 2ded. (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y., and Lakowicz, J. R. Principles of Fluorescence Spectroscopy, NewYork: Plenum Press (1983) for fluorescence techniques). Standardtechniques are used for chemical syntheses, chemical analyses, andbiological assays. As employed throughout the disclosure, the followingterms, unless otherwise indicated, shall be understood to have thefollowing meanings.

As used herein, the term “about” refers to a +/−10% variation from thenominal value. It is to be understood that such a variation is alwaysincluded in any given value provided herein, whether or not it isspecifically referred to.

The term “prostate-specific” as used herein with reference to an entityor moiety indicates that the entity/moiety, or a property of theentity/moiety, is selective to prostate cells when compared to othercell types. For example, a prostate specific entity/moiety can beselectively expressed by prostate cells, selectively associated withprostate cells, selectively activated by prostate cells, be capable ofselectively binding to prostate cells, or the like.

The term “prostate-specific activation sequence,” as used herein, refersto a sequence of amino acid residues which incorporates one or moreprostate-specific protease cleavage sites, which are selectively cleavedor hydrolysed by a prostate-specific protease.

The term “prostate-specific targeting domain,” as used herein, refers toa molecule such as a peptide ligand, toxin, or antibody, which iscapable of selectively binding to a prostate cell when compared to itsability to bind to other cell types.

The term “gene,” as used herein, refers to a segment of nucleic acidthat encodes an individual protein or RNA (also referred to as a “codingsequence” or “coding region”) together with associated regulatoryregions such as promoters, operators, terminators and the like, that maybe located upstream or downstream of the coding sequence.

The term “selectively hybridize,” as used herein, refers to the abilityof a nucleic acid to bind detectably and specifically to a secondnucleic acid. Polynucleotides, oligonucleotides and fragments thereofselectively hybridize to target nucleic acid strands under hybridizationand wash conditions that minimize appreciable amounts of detectablebinding to non-specific nucleic acids. High stringency conditions can beused to achieve selective hybridization conditions as known in the artand discussed herein. Typically, hybridization and washing conditionsare performed at high stringency according to conventional hybridizationprocedures. Washing conditions are typically 1-3×SSC, 0.1-1% SDS, 50-70°C. with a change of wash solution after about 5-30 minutes.

The terms “corresponding to” or “corresponds to” indicates that apolynucleotide sequence is identical to all or a portion of a referencepolynucleotide sequence. In contradistinction, the term “complementaryto” is used herein to indicate that the polynucleotide sequence isidentical to all or a portion of the complementary strand of a referencepolynucleotide sequence. For illustration, the nucleotide sequence“TATAC” corresponds to a reference sequence “TATAC” and is complementaryto a reference sequence “GTATA.”

The following terms are used herein to describe the sequencerelationships between two or more polynucleotides or two or morepolypeptides: “reference sequence,” “window of comparison,” “sequenceidentity,” “percent sequence identity,” and “substantial identity.” A“reference sequence” is a defined sequence used as a basis for asequence comparison; a reference sequence may be a subset of a largersequence, for example, as a segment of a full-length cDNA, gene orprotein sequence, or may comprise a complete cDNA, gene or proteinsequence. Generally, a reference polynucleotide sequence is at least 20nucleotides in length, and often at least 50 nucleotides in length. Areference polypeptide sequence is generally at least 7 amino acids inlength and often at least 17 amino acids in length.

A “window of comparison”, as used herein, refers to a conceptual segmentof the reference sequence of at least 15 contiguous nucleotide positionsor at least 5 contiguous amino acid positions over which a candidatesequence may be compared to the reference sequence and wherein theportion of the candidate sequence in the window of comparison maycomprise additions or deletions (i.e. gaps) of 20 percent or less ascompared to the reference sequence (which does not comprise additions ordeletions) for optimal alignment of the two sequences. The presentinvention contemplates various lengths for the window of comparison, upto and including the full length of either the reference or candidatesequence. Optimal alignment of sequences for aligning a comparisonwindow may be conducted using the local homology algorithm of Smith andWaterman (Adv. Appl. Math. (1981) 2:482), the homology alignmentalgorithm of Needleman and Wunsch (J. Mol. Biol. (1970) 48:443), thesearch for similarity method of Pearson and Lipman (Proc. Natl. Acad.Sci. (U.S.A.) (1988) 85:2444), using computerized implementations ofthese algorithms (such as GAP, BESTFIT, FASTA, and TFASTA in theWisconsin Genetics Software Package Release 7.0, Genetics ComputerGroup, 573 Science Dr., Madison, Wis.), using publicly availablecomputer software such as ALIGN or Megalign (DNASTAR), or by inspection.The best alignment (i.e. resulting in the highest percentage of identityover the comparison window) is then selected.

The term “sequence identity” means that two polynucleotide orpolypeptide sequences are identical (i.e. on a nucleotide-by-nucleotideor amino acid-by-amino acid basis) over the window of comparison.

The term “percent (%) sequence identity,” as used herein with respect toa reference sequence is defined as the percentage of nucleotide or aminoacid residues in a candidate sequence that are identical with theresidues in the reference polypeptide sequence over the window ofcomparison after optimal alignment of the sequences and introducinggaps, if necessary, to achieve the maximum percent sequence identity,without considering any conservative substitutions as part of thesequence identity.

The term “substantial identity” as used herein denotes a characteristicof a polynucleotide or polypeptide sequence, wherein the polynucleotideor polypeptide comprises a sequence that has at least 50% sequenceidentity as compared to a reference sequence over the window ofcomparison. Polynucleotide and polypeptide sequences which have at least60% sequence identity, at least 70% sequence identity, at least 80%sequence identity, or at least 90% sequence identity as compared to areference sequence over the window of comparison are also considered tohave substantial identity with the reference sequence.

The term “functional deletion” as used herein denotes a mutation,partial or complete deletion, insertion, or other variation made to agene sequence which renders that part of the gene sequencenon-functional. For example, functional deletion of a proaerolysin (PA)binding domain results in a decrease in the ability of PA to bind to andconcentrate on the cell membrane. This functional deletion can bereversed by inserting another functional binding domain intoproaerolysin, such as a prostate-specific targeting domain, for example,an LHRH peptide. Such reversal of a functional deletion is referred toherein as “functional replacement.” In another example, functionaldeletion of a native PA furin cleavage site results in a decrease in theability of PA to be cleaved and activated by furin, when compared to awild-type PA molecule.

The terms “therapy” and “treatment,” as used interchangeably herein,refer to an intervention performed with the intention of improving asubject's status. The improvement can be subjective or objective and isrelated to ameliorating the symptoms associated with, preventing thedevelopment of, or altering the pathology of a disease or disorder beingtreated. Thus, the terms therapy and treatment are used in the broadestsense, and include the prevention (prophylaxis), moderation, reduction,and curing of a disease or disorder at various stages. Preventingdeterioration of a subject's status is also encompassed by the term.Subjects in need of therapy/treatment thus include those already havingthe disease or disorder as well as those prone to, or at risk ofdeveloping, the disease or disorder and those in whom the disease ordisorder is to be prevented.

The term “ameliorate” includes the arrest, prevention, decrease, orimprovement in one or more the symptoms, signs, and features of thedisease or disorder being treated, both temporary and long-term.

The term “subject” or “patient” as used herein refers to an animal inneed of treatment.

The term “animal,” as used herein, refers to both human and non-humananimals, including, but not limited to, mammals, birds and fish.

Administration of the proteins or polypeptides of the invention “incombination with” one or more further therapeutic agents or additionaltreatment, is intended to include simultaneous (concurrent)administration and consecutive administration. Consecutiveadministration is intended to encompass administration of thetherapeutic agent(s) or additional treatment and the compound(s) of theinvention to the subject in various orders and via various routes.

The terms “antigen” and “antigenic material,” are used interchangeablyherein to refer to a molecule, molecules, a portion or portions of amolecule, or a combination of molecules, up to and including whole cellsand tissues, which are capable of inducing an immune response in ananimal. The antigenic material may comprise a single epitope orantigenic determinant or it may comprise a plurality of epitopes orantigenic determinants.

The term “immune response,” as used herein, refers to an alteration inthe reactivity of the immune system of an animal in response to anantigen or antigenic material and may involve antibody production,induction of cell-mediated immunity, complement activation and/ordevelopment of immunological tolerance.

The term “inhibit,” as used herein, means to decrease, reduce, slow-downor prevent.

“Binding pair” refers to two moieties (e.g. chemical or biochemical)that have an affinity for one another. Examples of binding pairs includehomo-dimers, hetero-dimers, antigen/antibodies, lectin/avidin, targetpolynucleotide/probe, oligonucleotide, antibody/anti-antibody,receptor/ligand, enzyme/ligand and the like. “One member of a bindingpair” refers to one moiety of the pair, such as an antigen or ligand.

“Isolated polynucleotide” refers to a polynucleotide of genomic, cDNA,or synthetic origin or some combination thereof, which by virtue of itsorigin the “isolated polynucleotide” (1) is not associated with the cellin which the “isolated polynucleotide” is found in nature, or (2) isoperably linked to a polynucleotide which it is not linked to in nature.

The term “polypeptide” is used herein as a generic term to refer to anamino acid sequence of at least 20 amino acids in length that can be awild-type (naturally-occurring) protein sequence, a fragment of awild-type protein sequence, a variant of a wild-type protein sequence, aderivative of a wild-type protein sequence, or an analogue of awild-type protein sequence. Hence, native protein sequences andfragments, variants, derivatives and analogues of native proteinsequences, as defined herein, are considered to be species of thepolypeptide genus.

The term “isolated polypeptide,” as used herein, refers to a polypeptidewhich by virtue of its origin is not associated with other polypeptideswith which it is normally associated with in nature, and/or is isolatedfrom the cell in which it normally occurs and/or is free of otherpolypeptides from the same cellular source and/or is expressed by a cellfrom a different species, and/or does not occur in nature.

“Naturally-occurring” or “native” as used herein, as applied to anobject, refers to the fact that an object can be found in nature. Forexample, a polypeptide or polynucleotide sequence that is present in anorganism (including viruses) that can be isolated from a source innature and which has not been intentionally modified by man in thelaboratory is naturally-occurring.

“Operably linked” refers to a juxtaposition wherein the components sodescribed are in a relationship permitting them to function in theirintended manner. A control sequence “operably linked” to a codingsequence is ligated in such a way that expression of the coding sequenceis achieved under conditions compatible with the control sequences.

“Control sequence” refers to polynucleotide sequences which arenecessary to effect the expression of coding and non-coding sequences towhich they are ligated. The nature of such control sequences differsdepending upon the host organism; in prokaryotes, such control sequencesgenerally include promoter, ribosomal binding site, and transcriptiontermination sequence; in eukaryotes, generally, such control sequencesinclude promoters and transcription termination sequences. The term“control sequences” is intended to include, at a minimum, componentswhose presence can influence expression, and can also include additionalcomponents whose presence is advantageous, for example, leader sequencesand fusion partner sequences.

“Polynucleotide” refers to a polymeric form of nucleotides of at least10 bases in length, either ribonucleotides or deoxynucleotides or amodified form of either type of nucleotide. The term includes single anddouble stranded forms of DNA or RNA.

“Polypeptide fragment” refers to a polypeptide that has anamino-terminal and/or carboxy-terminal deletion, but where the remainingamino acid sequence is usually identical to the corresponding positionsin the naturally-occurring sequence deduced, for example, from afull-length cDNA sequence. Fragments typically are at least 5, 6, 8 or10 amino acids long. In one embodiment, a fragment is at least 14 aminoacids long. In another embodiment, a fragment is at least 20 amino acidslong. In still another embodiment, a fragment is at least 50 amino acidslong. In yet another embodiment the fragment is at least 70 amino acidslong.

The term “label” or “labeled” refers to incorporation of a detectablemarker, e.g., by incorporation of a radiolabeled amino acid orattachment to a polypeptide of biotinyl moieties that can be detected bymarked avidin (e.g., streptavidin containing a fluorescent marker orenzymatic activity that can be detected by optical or colorimetricmethods). Various methods of labeling polypeptides and glycoproteins areknown in the art and may be used. Examples of labels for polypeptidesinclude, but are not limited to, the following: radioisotopes (e.g., ³H,¹⁴C, ³⁵S, ¹²⁵I, ¹³¹I), fluorescent labels (e.g., FITC, rhodamine,lanthanide phosphors), enzymatic labels (or reporter genes) (e.g.,horseradish peroxidase, β-galactosidase, (β-latamase, luciferase,alkaline phosphatase), chemiluminescent, biotinyl groups, predeterminedpolypeptide epitopes recognized by a secondary reporter (e.g., leucinezipper pair sequences, binding sites for secondary antibodies, metalbinding domains, epitope tags).

In some embodiments, labels are attached by spacer arms of variouslengths to reduce potential steric hindrance.

Naturally-occurring amino acids are identified throughout by theconventional three-letter or one-letter abbreviations indicated below,which are as generally accepted in the peptide art and are recommendedby the IUPAC-IUB commission in biochemical nomenclature:

TABLE 1 Amino acid codes 3-letter 1-letter Name code code Alanine Ala AArginine Arg R Asparagine Asn N Aspartic acid Asp D Cysteine Cys CGlutamic acid Glu E Glutamine Gln Q Glycine Gly G Histidine His HIsoleucine Ile I Alanine Leu L Arginine Lys K Asparagine Met M Asparticacid Phe F Cysteine Pro P Glutamic acid Ser S Glutamine Thr T GlycineTrp W Histidine Tyr Y Isoleucine Val V

The peptide sequences set out herein are written according to thegenerally accepted convention whereby the N-terminal amino acid is onthe left and the C-terminal amino acid is on the right. By convention,L-amino acids are represented by upper case letters and D-amino acids bylower case letters.

Modified Pore-Forming Proteins (MPPs)

The modified pore-forming proteins (MPPs) of the present invention arederived from naturally-occurring pore-forming proteins (nPPs), and havebeen modified to include one or more prostate-selective modificationssuch that they are capable of selectively killing normal prostate cellsrelative to cells from other normal tissues. By selective killing ofnormal prostate cells relative to cells from other normal tissues ismeant that the MPPs are capable of killing normal prostate cells moreeffectively than other types of normal cells such as, for example, lung,spleen, or blood cells. Suitable MPPs include those described in UnitedStates Patent Application No. 20040235095.

1. Naturally-Occurring Pore-Forming Proteins (nPPs)

Suitable mPPs from which the MPPs of the present invention can bederived include various bacterial toxins that are capable of formingpores or channels in the membrane of a target cell leading to celldeath. Suitable bacterial toxins include those that are produced asprotoxins and are subsequently activated by proteolytic cleavage as wellas those that are produced in an active from and do not requireadditional processing. In one embodiment, the nPPs are large cytotoxicproteins that are synthesized as protoxins which are activated byprotease cleavage at an activation sequence to form pores or channels inthe cell membrane of target cells, thus leading to rapid cytolytic celldeath. Suitable nPPs in accordance with this embodiment have thefollowing features: a pore-forming activity that is activated by removalof an inhibitory domain via protease cleavage, and the ability to bindto receptors that are present on cell membranes through one or morebinding domains. Numerous such nPPs have been cloned and recombinantforms produced (see, for example, Imagawa et al., FEMS. Microbiol. Lett.17:287-92, 1994; Meza et al. FEMS Microbiol. Lett. 145:333-9, 1996).

In one embodiment, the MPPs are derived from nPPs such as aerolysin oraerolysin-related polypeptides. Examples include, but are not limitedto, aerolysin homologues such as proaerolysin from Aeromonas hydrophila,Aeromonas trota and Aeromonas salmonicida, and alpha toxin fromClostridium septicum (Ballard et al., Infect. Immun. 63:340-4, 1995;Gordon et al. J. Biol. Chem. 274:27274-80, 1999; Genbank Accession No.S75954), as well as the following polypeptides: Bacillus anthracesprotective antigen, Vibrio cholerae VCC toxin, epsilon toxin fromClostridium perfringens, and Bacillus thuringiensis delta toxins(Genbank Accession No. D00117).

Proaerolysin (PA) polypeptides from the Aeromonas species noted abovehave been characterized. These polypeptides exhibit greater than 80%pairwise sequence identity between them (Parker et al., Progress inBiophysics & Molecular Biology 88 (2005) 91-142). Each of these PApolypeptides is an approximately 52 kDa protoxin with approximately 470amino acid residues. The cDNA sequence for wild-type PA from A.hydrophila is shown in SEQ ID NO: 1 (FIG. 7) and the corresponding aminoacid sequence of this wild-type PA is shown in SEQ ID NO:2 (FIG. 8). Thenucleotide and protein sequences for numerous naturally occurring nPPsare known in the art. Non-limiting examples are listed in the followingTable:

TABLE 2 Exemplary nPPs and corresponding GenBank ™ Accession NumbersNucleotide sequence Amino acid sequence (GenBank ™ (GenBank ™ nPPAccession No.) Accession No.) Aeromonas hydrophila Buckley AerA, BuckleyAerA aerolysin not corrected: M16495 corrected P09167 A. sobria Y00559CAA68642 proaerolysin¹ A. sobria X65046 CAA46182 hemolysin² A. trotaAF064068 AAC26217 proaerolysin³ A. salmonicida X65048 CAA46184hemolysin⁴, ¹Husslein et al., Mol. Microbiol. 2 (4), 507-517 (1988)²Hirono et al., Microb. Pathog. 13 (6), 433-446 (1992) ³Kahn et al.,Appl. Environ. Microbiol. 64 (7), 2473-2478 (1998) ⁴Hirono et al.,Microb. Pathog. 15 (4), 269-282 (1993)

The A. hydrophila PA protein includes a binding domain (approximatelyamino acids 1-83 of SEQ ID NO: 2) in what is known as the small lobe ofthe polypeptide and referred to herein as the small lobe binding domain(SBD), and a C-terminal inhibitory peptide (CIP) domain (approximatelyamino acids 427-470 of SEQ ID NO: 2) that is removed by proteasecleavage at an activation sequence to activate PA. Cleavage at theactivation sequence to remove the CIP domain can be carried out by anumber of ubiquitous proteases including furin and trypsin. The aminoacid residues from approximately 84-426 of SEQ ID NO: 2 are known as thelarge lobe of the PA polypeptide, and contain a toxin domain and otherfunctional domains, including a second binding domain, referred toherein as the large lobe binding domain (LBD). The cDNA sequence forwild-type A. hydrophila PA is shown in SEQ ID NO: 1.

Alpha toxin from C. septicum is considered to be a homologue ofproaerolysin based on significant sequence identity and othersimilarities (Parker et al., supra). Alpha toxin is secreted as a 46,450Da protoxin (approximately 443 amino acids) that is activated byprotease cleavage at an activation sequence to remove a C-terminalinhibitory peptide (CIP) domain, and it also binds toglycosyl-phosphatidylinositol (GPI)-anchored proteins. Alpha toxin,however, does not have a region corresponding to the small lobe of PA.Activation of this polypeptide occurs by protease cleavage at a furincleavage site (Gordon et al., Infect. Immun. 65:4130-4, 1997). Anexample of a Clostridium septicum alpha toxin nucleic acid sequence isprovided in GenBank™ Accession No. S75954 (SEQ ID NO:73, FIG. 40), andan example of a Clostridium septicum alpha toxin protein sequence isprovided in GenBank™ Accession No. AAB32892 (SEQ ID NO:74, FIG. 41).Based on the sequence homology, alpha toxin is thought to have a similarstructure and similar ability to bind to GPI-anchored proteins.

The activation sequence of Bacillus thuringiensis delta-toxin is cleavedby proteases in the midgut of certain insects to produce activeendotoxin (Miranda et al., Insect Biochem. Mol. Biol. 31:1155-63, 2001).The structure of this endotoxin has been solved and shown to consist ofthree domains, a channel-forming domain, a binding domain, and astabilizing domain.

In one embodiment, the MPPs according to the present invention arederived from proaerolysin polypeptides. In a further embodiment, theMPPs are derived from proaerolysin polypeptides from A. hydrophila. Inanother embodiment of the invention, the MPPs are derived from alphatoxin polypeptides.

In another embodiment, the MPPs are derived from nPPs that do notrequire protease cleavage for activation, and thus do not have anactivation sequence. These nPPs can be modified to insert aprostate-specific protease cleavage site into the nPP resulting in anMPP that is capable of being selectively activated to kill prostatecells. Examples of such nPPs include Staphylococcus aureus a hemolysin.In the case of this nPP, an activation sequence can be inserted inot thecenter of the pore-forming domain as known in the art (Panchal et al.,(1996) Nat. Biotech. 14:852-856).

The present invention further includes MPPs that are derived frombiologically active fragments of nPPs. Biologically active fragments ofnPPs are those that are capable of forming pores and killing cells.Suitable fragments include those that are capable of being activated toform pores in target cells by removal of a CIP domain. For example, inthe case of PA, a suitable fragment would be one that comprised abinding domain of the protein as well as the CIP domain and activationsequence. Thus, in one embodiment of the invention, the MPP is derivedfrom a fragment of proaerolysin that includes a binding domain, the CIPdomain and the activation sequence. In another embodiment, the MPP isderived from a fragment of proaerolysin that comprises the bindingdomain, the activation sequence, but only part of a CIP domain.

2. Prostate-Specific Modifications

In accordance with the present invention, the selected nPP is modifiedto form a MPP by inclusion of one or more prostate-specificmodifications. Prostate-specific modifications contemplated by thepresent invention include incorporation of a prostate-specificactivation sequence and/or functional deletion (including functionalreplacement) of one or more binding domains, and/or addition of aprostate-specific targeting domain.

In one embodiment, the MPPs according to the present invention comprisea prostate-specific activation sequence that allows for selectiveactivation of the MPPs in prostate cells. A prostate-specific activationsequence may be generated by modification of the naturally-occurringactivation sequence of a nPP, or it may be generated by the addition ofa prostate-specific activation sequence to a nPP that does not have anaturally-occurring activation sequence. In another embodiment, the MPPscomprise a prostate-specific activation sequence and one or moreprostate-specific targeting domains. In another embodiment, the MPPscomprise a prostate-specific activation sequence and a modification tothe SBD. In another embodiment, the MPPs comprise a prostate-specificactivation sequence and a modification to the LBD.

In one embodiment, the MPPs according to the present invention compriseone or more prostate-specific targeting domains that allow for selectiveactivation of the MPPs in prostate cells. In another embodiment, theMPPs comprise one or more prostate-specific targeting domain and amodification to the SBD. In another embodiment, the MPPs comprise aprostate-specific targeting domain and a modification to the LBD.

In still another embodiment, the MPPs comprise a prostate-specificactivation sequence, one or more prostate-specific targeting domain anda modification to the LBD. In another embodiment, the MPPs comprise aprostate-specific activation sequence, one or more prostate-specifictargeting domains, and a modification to the SBD.

In one embodiment, the MPP comprises a prostate-specific activationsequence and one or more modifications to the native binding domain. Inanother embodiment, the MPP comprises a prostate-specific targetingdomain and one or more modifications to the native binding domain. Instill another embodiment, the MPP comprises a prostate-specificactivation sequence, a prostate-specific targeting domain, and one ormore modifications to the native binding domain.

Representative, non-limiting examples of combinations ofprostate-specific modifications that can be made to proaerolysin areshown in FIGS. 4, 5, and 6.

Modification of Activation Sequence

As indicated above, a nPP can be modified to incorporate aprostate-specific activation sequence by modification of the naturallyoccurring activation sequence to provide a prostate-specific activationsequence, or a prostate-specific activation sequence can be added to annPP that does not have a naturally occurring activation sequence. Aprostate-specific activation sequence is accordance with the presentinvention is a sequence of amino acids that incorporates one or moreprostate-specific protease cleavage sites. A prostate-specific proteasecleavage site is a sequence of amino acids which is recognized andselectively and efficiently hydrolyzed (cleaved) by a prostate-specificprotease. In one embodiment, a prostate-specific protease is a proteasethat is expressed at higher levels in prostate cells than in other celltypes. Examples of prostate-specific proteases include, but are notlimited to: PSA (prostate-specific antigen), PSMA (prostate-specificmembrane antigen), and HK2 (human glandular kallikrein 2) cleavagesequences. Numerous examples of cleavage sites recognized by theseprostate-specific proteases are known in the art and will be describedfurther below.

Modifications to the naturally-occurring activation sequence to providea prostate-specific protease activation sequence may be achieved as isknown in the art. Modification of the naturally occurring activationsequence results in functional deletion of the native activationsequence. Functional deletion can be achieved by mutation, partial orcomplete deletion, insertion, or other variation made to the naturallyoccurring activation sequence that renders it inactive. In oneembodiment, the naturally-occurring activation sequence of the nPP isfunctionally deleted by insertion of a prostate-specific activationsequence. In another embodiment, functional deletion of the naturallyoccurring activation sequence is achieved via mutations in one or moreamino acid residues of the native activation sequence which produce aprostate-specific activation sequence. In an alternate embodiment, thenaturally occurring activation sequence of the nPP is functionallydeleted by replacing the native protease cleavage site of the activationsequence with a prostate-specific protease cleavage site.

In one embodiment, the one or more prostate-specific protease cleavagesites functionally replace the native protease cleavage site of the MPP.For example, a prostate-specific protease cleavage site can functionallyreplace the native furin cleavage site of PA (see FIG. 4B). Thisreplacement results in a MPP that becomes cytolytically active in thepresence of an enzymatically active prostate-specific protease, such asPSA, PSMA, or HK2. Suitable PSA, PSMA, or HK2 cleavage sites are knownin the art and are described below.

In another embodiment of the invention, the MPPs according to thepresent invention can be generated by deleting the native proteasecleavage site of the nPP and inserting a prostate-specific activationsequence. For example the furin cleavage site of PA (amino acids 427-432of SEQ ID NO: 2) can be deleted and a prostate-specific proteasecleavage site, such as a PSA cleavage site, inserted (see FIG. 4B).

In a further embodiment, the native protease cleavage site of the nPP ismutated such that it is no longer functional and a prostate-specificactivation sequence is inserted within the mutated protease cleavagesite, or added to the N- or C-terminus of the native protease cleavagesite. For example, the furin cleavage site of PA can be mutated and aprostate-specific protease cleavage site, such as a PSA cleavage site,inserted within, or added to the N- or C-terminus of the mutated furinsite (see FIG. 4C).

In still another embodiment, a prostate-specific activation sequence isadded to an nPP that does not have a naturally occurring activationsequence. For example, Staphylococcus aureus α-hemolysin, which does notrequire protease cleavage in order to be activated to kill cells, may beengineered to include one or more prostate-specific protease cleavagesites, thus rendering it capable of being selectively activated to killprostate cells.

Prostate-Specific Cleavage Sites

As noted above, various prostate-specific proteases and the proteasecleavage sites they recognize are known in the art. Examples include,but are not limited to, PSA, PSMA and HK2.

In one embodiment, the MPP is modified to include a prostate-specificactivation sequence that includes a PSA-specific cleavage site. APSA-specific cleavage site is a sequence of amino acids which isrecognized and selectively and efficiently hydrolyzed (cleaved) byprostate specific antigen (PSA). PSA is a serine protease with theability to recognize and hydrolyze specific peptide sequences. It issecreted by prostate cells in an enzymatically active form and becomesinactivated upon entering the circulation. Since neither blood nornormal tissue other than the prostate contains enzymatically active PSA,the proteolytic activity of PSA can be used to activate MPPs at theprostate gland. Various PSA-specific cleavage sites are known in theart. Examples, include, but are not limited to, those shown in SEQ IDNOs: 5, 8, 11, and 14-21, and those disclosed in U.S. Pat. Nos.5,866,679, 5,948,750, 5,998,362, 6,265,540, 6,368,598, and 6,391,305. Inone embodiment, the MPP has an activation sequence that includes the PSAcleavage site shown in SEQ ID NO: 5. Additional PSA-specific cleavagesites are known, based on the PSA-cleavage map of human seminal proteinssemenogelin I and II, and a cellulose membrane based assay (see Table 3and Denmeade et al., Cancer Res., 57:4924-30, 1997) and can be used toproduce the modified MPPs according to the present invention. Forexample, the MPPs according to the present invention can be modified toinclude one of the PSA-cleavage sites as shown in Table 3, which cansubstitute for the wild-type furin protease activation site ofproaerolysin (amino acids 427-432 of SEQ ID NO: 2), as is known in theart.

In one embodiment, the MPP has an amino acid sequence of any one of SEQID NOs: 3, 4, 6, 7, 9, 10, 12, 13, and 24, which include an activationsequence containing a PSA cleavage site.

TABLE 3 PSA substrates (PSA cleavage sites) and kinetics of PSAhydrolysis.* PSA substrate (SEQ ID NO) K_(m) (μM) K_(cat) (s⁻¹)K_(cat)/K_(m) (s⁻¹ M⁻¹) KGISSQY (15) 160 0.043 270 SRKSQQY (16) 90 0.023260 ATKSKQH (17) 1310 0.0091 6.9 KGLSSQC (18) 300 0.0017 5.6 LGGSSQL(19) 900 0.0037 4.1 EHSSKLQ (20) 1165 0.012 10.6 HSSKLQ (5) 470 0.01123.6 SKLQ (21) 813 0.020 24.6 *Peptides were fluorescently labeled(aminomethyl coumarin). Assays were performed in 50 mM Tris, 0.1 M NaCl,pH 7.8.

In another embodiment, the MPP comprises a prostate-specific activationsequence that includes a PSMA-specific cleavage site. Examples ofsuitable PSMA-specific cleavage sites are known in the art and can befound, for example, in International Publication No. WO 02/43773. Ingeneral terms, a PSMA cleavage site includes at least the dipeptideX₁X₂. The dipeptide contains the amino acids Glu or Asp at position X₁.X₂ can be Glu, Asp, Gln, or Asn. Tripeptides X₁X₂X₃ are also suitable,with X₁ and X₂ defined as before, with X₃ as Glu, Asp, Gln or Asn.Tetrapeptides X₁X₂X₃X₄ are also suitable, with X₁₋₃ defined as above,and with X₄ as Glu, Asp, Gln or Asn. Pentapeptides X₁X₂X₃X₄X₅ are alsosuitable, with X₁₋₄ defined as above, and with X₅ as Glu, Asp, Gln orAsn. Hexapeptides X₁X₂X₃X₄X₅X₆ are also suitable, with X₁₋₅ defined asabove, and with X₆ as Glu, Asp, Gln or Asn. Further peptides of longersequence length can be constructed in similar fashion. Generally, thepeptides are of the following sequence: X₁ . . . X_(n), where n is 2 to30, 2 to 20, 2 to 15, or 2 to 6, where X₁ is Glu, Asp, Gln or Asn. Inone embodiment, X₁ is Glu or Asp, and X₂-X_(n) are independentlyselected from Glu, Asp, Gln and Asn. Other possible peptide sequencesare as above, except that X₂-X_(n-1) are independently selected fromGlu, and Asp, and X_(n) is independently selected from Glu, Asp, Gln andAsn. Examples of PSMA cleavage sites are Asp-Glu, Asp-Asp, Asp-Asn,Asp-Gln, Glu-Glu-Glu, Glu-Asp-Glu, Asp-Glu-Glu, Glu-Glu-Asp,Glu-Asp-Asp, Asp-Glu-Asp, Asp-Asp-Glu, Asp-Asp-Asp, Glu-Glu-Gln,Glu-Asp-Gln, Asp-Glu-Gln, Glu-Glu-Asn, Glu-Asp-Asn, Asp-Glu-Asn,Asp-Asp-Gln, and Asp-Asp-Asn.

In an additional embodiment, the MPP comprises a prostate-specificactivation sequence that includes an HK2-specific cleavage site.Examples of HK2-specific cleavage sites are also known in the art anddescribed, for example, in International Publication No. WO01/09165. Thecleavage site recognized by HK2 is flanked by at least an amino acidsequence X₄X₃X₂X₁. This amino acid sequence contains the amino acidarginine, histidine or lysine at position X₁. X₂ can be arginine,phenylalanine, lysine, or histidine. X₃ can be lysine, serine, alanine,histidine or glutamine. X₄ can be from 0 to 20 further amino acids, andcan be at least two further amino acids. In an embodiment, the HK2cleavage site includes a sequence for X₄ that is substantially identicalto the 20 amino acids in the wild type semenogelin I or semenogelin IIsequence that are the from fourth to twenty fourth amino acids to theN-terminal side of recognized semenogelin cleavage sites. The amino acidsequence can further comprise X₁, which is linked to the carboxyterminus of X₁ to create the amino acid sequence X₄X₃X₂X₁X₁. X₁ is up toa further 10 amino acids, and can include various amino acids. X₁ mayhave a leucine, alanine or serine linked to the carboxy terminus of X₁.X₁ can include L- or D-amino acids. The HK2 cleavage site is located atthe carboxy terminal side of X₁.

Examples of HK2 cleavage sites are shown in Table 4 (Note that thesymbol][denotes an Hk2 cleavage site):

TABLE 4 Exemplary HK2 Cleavage sites Lys-Arg-Arg ][ SEQ ID NO: 32Ser-Arg-Arg ][ Leu SEQ ID NO: 53 Ser-Arg-Arg ][ SEQ ID NO: 33Ala-Arg-Arg ][ Leu SEQ ID NO: 54 Ala-Arg-Arg ][ SEQ ID NO: 34Ala-Arg-Arg ][ Ser SEQ ID NO: 55 His-Arg-Arg ][ SEQ ID NO: 35His-Arg-Arg ][ Ala SEQ ID NO: 56 Gln-Arg-Arg ][ SEQ ID NO: 36Gln-Arg-Arg ][ Leu SEQ ID NO: 57 Ala-Phe-Arg ][ SEQ ID NO: 37Ala-Phe-Arg ][ Leu SEQ ID NO: 58 Ala-Gln-Arg ][ SEQ ID NO: 38Ala-Gln-Arg ][ Leu SEQ ID NO: 59 Ala-Lys-Arg ][ SEQ ID NO: 39Ala-Lys-Arg ][ Leu SEQ ID NO: 60 Ala-Arg-Lys ][ SEQ ID NO: 40Ala-Arg-Lys ][ Leu SEQ ID NO: 61 Ala-His-Arg ][ SEQ ID NO: 41Ala-His-Arg ][ Leu SEQ ID NO: 62 Gln-Lys-Arg-Arg ][ SEQ ID NO: 42His-Ala-Gln-Lys-Arg-Arg ][ Leu SEQ ID NO: 63 Lys-Ser-Arg-Arg ][ SEQ IDNO: 43 Gly-Gly-Lys-Ser-Arg-Arg ][ Leu SEQ ID NO: 64 Ala-Lys-Arg-Arg ][SEQ ID NO: 44 His-Glu-Gln-Lys-Arg-Arg ][ Leu SEQ ID NO: 65Lys-Lys-Arg-Arg ][ SEQ ID NO: 45 His-Glu-Ala-Lys-Arg-Arg ][ Leu SEQ IDNO: 66 His-Lys-Arg-Arg ][ SEQ ID NO: 46 Gly-Gly-Gln-Lys-Arg-Arg ][ LeuSEQ ID NO: 67 Lys-Ala-Phe-Arg ][ SEQ ID NO: 47 His-Glu-Gln-Lys-Arg-Arg][ Ala SEQ ID NO: 68 Lys-Ala-Gln-Arg ][ SEQ ID NO: 48Gly-Gly-Ala-Lys-Arg-Arg ][ Leu SEQ ID NO: 69 Lys-Ala-Lys-Arg ][ SEQ IDNO: 49 His-Glu-Gln-Lys-Arg-Arg ][Ser SEQ ID NO: 70 Lys-Ala-Arg-Lys ][SEQ ID NO: 50 Gly-Gly-Lys-Lys-Arg-Arg ][ Leu SEQ ID NO: 71Lys-Ala-His-Arg ][ SEQ ID NO: 51 Gly-Gly-His-Lys-Arg-Arg ][ Leu SEQ IDNO: 72 Lys-Arg-Arg ][ SEQ ID NO: 52 Leu

Addition of Prostate-Specific Targeting Domain

In one embodiment of the invention, the MPPs comprise one or moreprostate-specific targeting domains to allow selective targeting ofprostate cells. The prostate-specific targeting domain is capable ofdirecting the MPP to the prostate cell, where the MPP can be activatedand subsequently kill the prostate cell. The targeting domain can belocated at the N- or C-terminus of the MPP, or both. Alternatively, thetargeting domain can located at another region of the MPP, as long as itdoes not interfere with the pore-forming activity of the MPP.

Examples of suitable prostate-specific targeting domains include, butare not limited to molecules such as a peptide ligand, toxin, orantibody, which have a higher specificity for prostate cells than forother cell types. In one embodiment, a prostate tissue specific bindingdomain has a lower K_(D) in prostate tissue or cells than in other celltypes, (i.e. binds selectively to prostate tissues as compared to othernormal tissues), for example at least a 10-fold lower K_(D), such as anat least 20-, 50-, 75-, 100- or even 200-fold lower K_(D). Suchmolecules can be used to target a MPP to the prostate. Examples include,but are not limited to: antibodies which recognize proteins that arerelatively prostate-specific such as PSA, PSMA, HK2, prostasin, andhepsin; ligands which have prostate-specific receptors such as naturaland synthetic luteinizing hormone releasing hormone (LHRH); andendothelin (binding to cognate endothelin receptor).

In one embodiment of the invention, addition of the prostate-specifictargeting domain results in functional deletion of the native bindingdomain of the nPP. In another embodiment, the native non-specificGPI-anchor protein binding domain of proaerolysin is functionallydeleted and replaced with a prostate-specific targeting domain. Specificexamples of MPPs derived from proaerolysin that have a functionallydeleted native binding domain are depicted in FIGS. 4D and 5B. Examplesof MPPs derived from proaerolysin that include a prostate-specifictargeting domain which functionally substitutes for the nativeproaerolysin binding domain are shown in FIGS. 4E, 5A, 5C and 5D and6A-6D.

One or more prostate tissue-specific binding domains can be linked toone or more amino acids of the MPPs, but ideally, do not interferesignificantly with the ability to form pores in cell membranes, or,where applicable, with the ability of the MPP to be activated by aprostate-specific protease such as PSA. Methods of conjugating proteinsor peptides to MPPs are known in the art and include for example,changing the N-terminal amino acid of the protein to be modified to aCys or other amino acid before attaching the prostate-tissue specificbinding domain, to assist in linking the prostate-tissue specificbinding domain to the MPP.

In one embodiment, prostate tissue specific binding domains are linkedor inserted at the N- and/or C-terminus of an MPP derived fromproaerolysin (for example, see FIGS. 4E and 5C). In some examples, thenative binding domain of proaerolysin is deleted (i.e. amino acids 1-83of SEQ ID NO: 2 or 4), such that attachment or linking of a prostatetissue specific binding domain to the N-terminus results in attachmentto amino acid 84 of SEQ ID NO: 2 or 4 (for example, see FIGS. 5C and6C). In other examples, smaller deletions or point mutations areintroduced into the native binding domain of proaerolysin, such thatattachment or linking of a prostate tissue specific binding domain tothe N-terminus results in attachment to amino acid 1 of SEQ ID NO: 2 or4 (or whichever amino acid is N-terminal following functional deletionof the native proaerolysin binding domain) (for example, see FIGS. 4Eand 5D).

Antibodies as Prostate-Specific Targeting Domains

In one embodiment, the prostate specific targeting domain is an antibodyor antibody fragment that specifically binds to an antigen that isassociated with prostate cells, thus targeting the MPP to prostatecells. Antigens associated with prostate cells that may be specificallybound by such prostate-specific targeting domains include PSA, and PSMAand the LHRH receptor, the expression of which is elevated in prostatecells. Antibodies can be attached to the N- or C-terminus of the MPPusing gene fusion methods well known in the art (for example seeDebinski and Pastan, Clin. Cancer Res. 1:1015-22, 1995). Alternatively,antibodies can be attached to an MPP by covalent crosslinking (forexample see Woo et al., Arch. Pharm. Res. 22(5):459-63, 1999 andDebinski and Pastan, Clin. Cancer Res. 1(9):1015-22, 1995). Crosslinkingcan be non-specific, for example by using ahomobifunctional-lysine-reactive crosslinking agent, or it can bespecific, for example by using a crosslinking agent that reacts withamino groups on the antibody and with cysteine residues located in theMPP. In one embodiment, proaerolysin amino acids such as amino acidsCys19, Cys75, Cys159, and/or Cys164 of SEQ ID NO: 2 can be used tocrosslink antibodies to the modified proaerolysin molecule. For example,the antibody could replace the native binding domain of the nPP to bemodified, or the antibody could be added to an MPP already havingmutations in the native binding domain. Such an MPP can also include aprostate-specific activation sequence to increase specificity. In oneembodiment, the antibody is a single chain antibody to PSMA fused to thetoxin domain of PA.

Suitable antibodies include intact antibodies as well as antibodyfragments such as, for example, (i) an Fab fragment consisting of theVL, VH, CL and CH1 domains; (ii) an Fd fragment consisting of the VH andCH1 domains; (iii) an Fv fragment consisting of the VL and VH domains ofa single arm of an antibody, (iv) a dAb fragment (Ward et al., Nature341:544-6, 1989) which consists of a VH domain; (v) an isolatedcomplementarity determining region (CDR); and (vi) an F(ab′)2 fragment,a bivalent fragment comprising two Fab fragments linked by a disulfidebridge at the hinge region. Suitable antibodies include single chain Fvantibodies, which are prepared by recombinant methods resulting in thetwo domains of an Fv fragment being linked via a synthetic linker (Birdet al. Science 242:423-6, 1988; and Huston et al., Proc. Natl. Acad.Sci. 85:5879-83, 1988), and camelized antibodies (for example see Tanhaet al., J. Biol. Chem. 276:24774-80, 2001).

In another embodiment, the antibody fragments are capable ofcrosslinking their target antigen, e.g., bivalent fragments such asF(ab′)2 fragments. Alternatively, an antibody fragment which does notitself crosslink its target antigen (e.g., a Fab fragment) can be usedin conjunction with a secondary antibody which serves to crosslink theantibody fragment, thereby crosslinking the target antigen. Antibodiescan be fragmented using conventional techniques and the fragmentsscreened for utility in the same manner as described for wholeantibodies, and as is known in the art. An antibody is further intendedto include nanobodies, and bispecific and chimeric molecules thatspecifically bind the target antigen.

“Specifically binds,” when used in reference to an antibody, refers tothe ability of individual antibodies to specifically immunoreact with aspecific antigen. The binding is a non-random binding reaction betweenan antibody molecule and an antigenic determinant of the antigen. Thedesired binding specificity is typically determined from the referencepoint of the ability of the antibody to differentially bind the specificand an unrelated antigen, and therefore distinguish between twodifferent antigens, particularly where the two antigens have uniqueepitopes. An antibody that specifically binds to a particular epitope isreferred to as a “specific antibody”.

Small Peptide Ligands as Prostate-Specific Targeting Domains

In one embodiment, the prostate-specific targeting domain is a smallpeptide ligand that binds to its cognate prostate-specific receptorexpressed on the membrane of prostate cells. Examples include, but arenot limited to, natural and synthetic luteinizing hormone releasinghormone (LHRH) agonist peptides (for example see Genbank Accession No.CAA25526 and SEQ ID NOS: 22 and 23), which bind with high affinity toLHRH receptors, and peptides that can bind selectively to PSMA. LHRHreceptors are displayed by prostate cells, and only a few other cells.This differential expression provides binding specificity.

Small peptide ligands may be modified as is known in the art in order tofacilitate their attachment to the MPP. For example, certain residues ofLHRH, such as the Gly at the 6th position (Gly6), can be substitutedwithout compromising receptor binding affinity (Janaky et al., Proc.Natl. Acad. Sci. USA 89:972-6, 1992; Nechushtan et al., J. Biol. Chem.,272:11597-603, 1997). Therefore, an MPP (in which the native bindingdomain is functionally deleted) can be produced which is covalentlycoupled to purified LHRH D-Lys6 (at the epsilon amine of this lysine).

LHRH D-Lys6 (SEQ ID NO: 23) can be attached at various positions withinan nPP to provide an MPP having a prostate-specific targeting domain. Asnoted above, attachment of the small peptide ligand will notsignificantly interfere with the ability of the toxin to insert into themembrane to form a pore. For example, the epsilon amine of the D-Lys6analog can be coupled to the amino terminus of the MPP using methodsknown in the art such as for example via a dicarboxylic acid linker.Activation of the MPP by cleavage of the activation sequence will resultin release of the C-terminal inhibitory portion while the toxin remainsbound to the LHRH receptor.

Alternatively or in addition, the small peptide ligand can be coupleddirectly to the C-terminus of the MPP. For example, the epsilon amine ofthe D-Lys6 analog of LHRH can be coupled directly to the C-terminalcarboxyl of the MPP by the addition of a Cys to the C-terminus of theMPP, then crosslinking this Cys to the epsilon amine of the D-Lys6analog of LHRH. This coupling will produce an MPP in which the LHRHpeptide is attached to the C-terminal inhibitory domain. Activation ofthe MPP by cleavage of the activation sequence will liberate the MPP andleave the inhibitory fragment bound to the LHRH receptor. In addition,recombinant fusion proteins can be produced in which modified LHRHpeptides are fused to both the N- and C-terminus of the MPP.

It is also contemplated that the small peptide ligand may be attached toan MPP via a disulfide bridge. For example, a cysteine residue isintroduced into the 6th position of the LHRH peptide and the peptideattached to an MPP via a disulfide bridge. The cysteine with which thepeptide forms a disulphide bridge can be present in the native nPPsequence or the nPP can be mutated to include a cysteine residue. In oneembodiment, an MPP derived from PA can have a cysteine residueintroduced, for example at amino acids 215 and/or 300 of SEQ ID NO: 2,wherein amino acid 215 and/or 300 has been mutated to a cysteine.

In another embodiment, a recombinant protein is produced in which LHRHpeptide is fused to the amino terminus of the MPP.

Alternatively or in addition, an MPP may be produced by attaching orlinking one or more prostate-specific targeting domains to other aminoacids of the MPP. For example, for MPPs derived from proaerolysin, aminoacids such as amino acid 215 or 300 of SEQ ID NO: 2 or 4 (for example,see FIGS. 5A, 5D, 6B and 6D) may be used to attach the one or moreprostate specific targeting domains. In some examples, a Cys amino acidreplaces the native amino acid at that position. For example, thefollowing changes can be made to SEQ ID NO: 2 or 4: Tyr215Cys orAla300Cys. Alternatively, cysteine residues present in the nativesequence of the nPP can be utilized. For MPPs derived from proaerolysin,amino acids such as amino acids Cys19, Cys75, Cys159, and/or Cys164 ofSEQ ID NO: 2, are suitable for this purpose.

In one embodiment the MPP is derived from proaerolysin and has asequence selected from SEQ ID NOs:24 and 25, which comprises LHRH as aprostate-specific targeting domain.

Modifications to the Native Binding Domain of MPPs

MPPs according to the present invention are derived from nPPs thatcomprise one or more binding domains, as known in the art. In thecontext of the present invention, when an nPP comprises one bindingdomain, it is considered to be a “large lobe binding domain.” MPPsaccording to the present invention may comprise modifications to one ormore binding domains, as applicable. For example, native proaerolysinfrom Aeromonas species comprises two binding domains, a small lobebinding domain, and a large lobe binding domain. In contrast, nativealpha toxin from Clostridium septicum comprises only a large lobebinding domain. In one embodiment, modifications of the binding domainsinclude functional deletion of a binding domain. A functionally deletedbinding domain in an MPP results in an MPP that has an attenuatedability to bind to its cell surface receptor, yet still retainspore-forming ability. Functional deletions can be made by deleting ormutating one or more binding domains of an MPP. In one embodiment, theentire binding domain or portions thereof, may be deleted. In anadditional embodiment, insertion of heterologous sequences into thebinding domain may also be used to functionally delete the bindingdomain. Addition of these heterologous sequences may confer anadditional functionality to the MPP (i.e. functional replacement of thebinding domain). For example, addition of a heterologous sequence canresult in the addition of a region that can function as aprostate-specific targeting domain as described herein. In still anotherembodiment, point mutations to the amino acid sequence of the nativebinding domain of the nPP can also be made to decrease the ability ofthe binding domain to bind to its receptor. Further details regardingthese modifications are described below.

MPPs lacking a binding domain retain their cytolytic activity, but mayneed to be administered at higher doses to ensure concentration of thetoxin in the cell membrane. MPPs with functional deletions in thebinding domain may be prepared using methods known in the art. Thesemethods include the use of recombinant DNA technology as described inSambrook et al., supra. Alternatively, functional deletions of thebinding domain may also be achieved by direct modification of theprotein itself according to methods known in the art, such asproteolysis to generate fragments of the MPP, which can then bechemically linked together.

In one embodiment of the invention, the MPP is modified by functionaldeletion of its small lobe binding domain (SBD). Exemplary functionaldeletions of the SBD may be made in the A. hydrophila proaerolysinpolypeptide as follows. The entire SBD, corresponding to amino acid 1-83of SEQ ID NO:2 may be deleted, or portions of this region may bedeleted, for example amino acids 45-66 of SEQ ID NO:2. Alternatively,point mutations can be made as follows W45A, I47E, M57A, Y61A, K66Q(amino acid numbers refer to SEQ ID NO: 2 or SEQ ID NO:4) and asdescribed in Mackenzie et al. J. Biol. Chem. 274: 22604-22609, 1999. Aschematic diagram representing an example of an MPP with one or moremutations in a binding domain is shown in FIG. 4D, where * representsone or more mutations or deletions.

In one embodiment of the invention, the nPP is modified by functionaldeletion of its large lobe binding domain (LBD). Exemplary functionaldeletions of the LBD of proaerolysin (contained in approximately aminoacid residues 84-426 of SEQ ID NO:2) that may be made to provide MPPsare as follows. The entire LBD of proaerolysin may be deleted.Alternatively, in one embodiment of the invention, the MPP derived fromproaerolysin comprises one or more point mutations in the LBD to aminoacid residues Y162, W324, R323, R336, and/or W127. In another embodimentof the invention, the MPP derived from proaerolysin comprises one ormore point mutations at positions W127 and/or R336. In still anotherembodiment, the MPP derived from proaerolysin comprises the pointmutations Y162A and/or W324A. In a further embodiment the MPP derivedfrom proaerolysin comprises the point mutations R336A, R336c, and/orW127T. In another embodiment, MPPs comprise mutations to other residuesthat interact directly with the GPI-protein ligand.

Exemplary mutations to the LBD of MPPs derived from alpha toxin arenoted below and include at least one substituted amino acid in thereceptor binding domains of the alpha toxin which include amino acidresidues 53, 54, 62, 84-102, 259-274 and 309-315 of the sequence of thenative alpha toxin as shown in SEQ ID NO: 33. In one embodiment of theinvention, MPPs derived from alpha toxin include mutations to one ormore of the following residues: W85, Y128, R292, Y293, and 8305.

Further Modifications of MPPs

The present invention contemplates further modification of MPPs that donot affect the ability of the MPPs to selectively target prostate cells.Such modifications include amino acid substitutions, insertions ordeletions, modifications to reduce antigenicity, and modifications toenhance the stability or improve the pharmacokinetics of the MPPs. Inone embodiment, further modifications to MPPs result in a polypeptidethat differs by only a small number of amino acids from the MPP. Suchmodifications include deletions (for example of 1-3 or more aminoacids), insertions (for example of 1-3 or more residues), orsubstitutions that do not interfere with the ability of the MPPs toselectively target and kill normal prostate cells. In one embodiment,further modifications to the MPPs result in a polypeptide that retainsat least 70%, 80%, 85%, 90%, 95%, 98%, or greater sequence identity tothe MPP and maintains the ability of the MPP to selectively target andkill normal prostate cells.

MPPs may be modified by substitution whereby at least one residue in theamino acid sequence has been removed and a different residue inserted inits place. In one embodiment, the substitution is a conservativesubstitution. A conservative substitution is one in which one or moreamino acids (for example 2, 5 or 10 residues) are substituted with aminoacid residues having similar biochemical properties. Typically,conservative substitutions have little to no impact on the activity of aresulting polypeptide. For example, ideally, an MPP including one ormore conservative substitutions retains the activity of thecorresponding nPP. Examples of amino acids which may be substituted foran original amino acid in a protein and which are regarded asconservative substitutions include: Ser for Ala; Lys for Arg; Gln or Hisfor Asn; Glu for Asp; Ser for Cys; Asn for Gln; Asp for Glu; Pro forGly; Asn or Gln for His; Leu or Val for Ile; Ile or Val for Leu; Arg orGln for Lys; Leu or Ile for Met; Met, Leu or Tyr for Phe; Thr for Ser;Ser for Thr; Tyr for Trp; Trp or Phe for Tyr; and Ile or Leu for Val.

An MPP can be modified to include one or more conservative substitutionsby manipulating the nucleotide sequence that encodes that polypeptideusing, for example, standard procedures such as site-directedmutagenesis or PCR. Further information about conservative substitutionscan be found in, among other locations, Ben-Bassat et al., (J.Bacteriol. 169:751-7, 1987), O'Regan et al., (Gene 77:237-51, 1989),Sahin-Toth et al., (Protein Sci. 3:240-7, 1994), Hochuli et al.,(Bio/Technology 6:1321-5, 1988), WO 00/67796 (Curd et al.) and instandard textbooks of genetics and molecular biology.

In another embodiment the substitution is a permissive substitution.Permissive substitutions are non-conservative amino acid substitutions,but also do not significantly alter MPP activity. An example issubstitution of Cys for Ala at position 300 of SEQ ID NO: 2 or 4 in aproaerolysin polypeptide.

In one embodiment, MPPs are modified to include 1 or more amino acidsubstitutions of single residues. In another embodiment, the MPPs aremodified to include 1 amino acid substitution. In another embodiment,the MPPs are modified to include from about 2 to about 10 amino acidsubstitutions. In another embodiment, the MPPs are modified to includeabout 3 to about 5 amino acid substitutions.

Non-limiting examples of further modifications to MPPs derived fromproaerolysin are listed in Table 5.

TABLE 5 Exemplary single mutations of MPPs derived from a nativeproaerolysin polypeptide H107N G202C G251C T284C H341N K22C H121N W203CE252C V285C W127T T253S V293C K361C N459C C164S D216C T253C K294C K369QQ254C K294Q W371L D372N I445C Y135A R220Q E296C K299C K349C Y135F K171CK238C W373L A418C K22C A300C S256C K309C H332N H186N P248C E258C I416CQ263C K198C L249C I259C G417C K114C C159S V201C V250C

Peptidomimetic and organomimetic embodiments are also contemplated,whereby the three-dimensional arrangement of the chemical constituentsof such peptido- and organomimetics mimic the three-dimensionalarrangement of the polypeptide backbone and component amino acid sidechains in the polypeptide, resulting in such peptido- and organomimeticsof an MPP which have the ability to lyse prostate cells. For computermodeling applications, a pharmacophore is an idealized,three-dimensional definition of the structural requirements forbiological activity. Peptido- and organomimetics can be designed to fiteach pharmacophore with current computer modeling software (usingcomputer assisted drug design or CADD). See Walters, “Computer-AssistedModeling of Drugs”, in Klegerman & Groves, eds., 1993, PharmaceuticalBiotechnology, Interpharm Press: Buffalo Grove, Ill., pp. 165-174 andPrinciples of Pharmacology (ed. Munson, 1995), chapter 102 for adescription of techniques used in CADD.

Other modifications that may be made to the MPPs include, for example,modifications to the carboxylic acid groups of the MPP, whethercarboxyl-terminal or side chain, in which these groups are in the formof a salt of a pharmaceutically-acceptable cation or esterified to forma C₁-C₁₆ ester, or converted to an amide of formula NR₁R₂ wherein R₁ andR₂ are each independently H or C₁-C₁₆ alkyl, or combined to form aheterocyclic ring, such as a 5- or 6-membered ring. Amino groups of thepolypeptide, whether amino-terminal or side chain, can be in the form ofa pharmaceutically-acceptable acid addition salt, such as the HCl, HBr,acetic, benzoic, toluene sulfonic, maleic, tartaric and other organicsalts, or may be modified to C₁-C₁₆ alkyl or dialkyl amino or furtherconverted to an amide.

Other modifications include conversion of hydroxyl groups of thepolypeptide side chain to C₁-C₁₆ alkoxy or to a C₁-C₁₆ ester usingwell-recognized techniques. Phenyl and phenolic rings of the polypeptideside chain can be substituted with one or more halogen atoms, such as F,Cl, Br or I, or with C₁-C₁₆ alkyl, C₁-C₁₆ alkoxy, carboxylic acids andesters thereof, or amides of such carboxylic acids. Methylene groups ofthe polypeptide side chains can be extended to homologous C₂-C₄alkylenes. Thiols can be protected with any one of a number ofwell-recognized protecting groups, such as acetamide groups. Thoseskilled in the art will also recognize methods for introducing cyclicstructures into the polypeptides described herein to select and provideconformational constraints to the structure that result in enhancedstability. For example, a carboxyl-terminal or amino-terminal cysteineresidue can be added to the polypeptide, so that when oxidized thepolypeptide will contain a disulfide bond, generating a cyclic peptide.Other peptide cyclizing methods include the formation of thioethers andcarboxyl- and amino-terminal amides and esters.

The present invention also contemplates further modifications to MPPs inwhich the MPPs are linked or immobilized to a surface, such as a bead.The bead can also include a prostate-specific ligand to enhancetargeting to a prostate cell. Immobilized refers to binding to asurface, such as a solid surface. A solid surface can be polymeric, suchas polystyrene or polypropylene. The solid surface may be in the form ofa bead. In one embodiment, the surface includes an immobilized MPP, andin other embodiments further includes one or more prostate-specificbinding ligands, such as LHRH peptide, PSMA antibody, and PSMA singlechain antibody. In another embodiment, the MPP is liberated from thebead once the bead reaches the prostate cell target. Methods ofimmobilizing peptides on a solid surface are known in the art and can befound in WO 94/29436, and U.S. Pat. No. 5,858,358.

The present invention further contemplates that the MPP can comprisefurther modifications intended to improve the pharmacokinetic propertiesof the molecule when administered to a subject. Various modifications toreduce immunogenicity and/or improve the half-life of therapeuticproteins are known in the art. For example, the MMPs can undergoglycosylation, isomerization, or deglycosylation according to standardmethods known in the art. Similarly, the MPP can be modified bynon-naturally occurring covalent modification for example by addition ofpolyethylene glycol moieties (pegylation) or lipidation. In oneembodiment, the MPPs of the invention are conjugated to polyethyleneglycol (PEGylated) to improve their pharmacokinetic profiles.Conjugation can be carried out by techniques known to those skilled inthe art (see, for example, Deckert et al., Int. J. Cancer 87: 382-390,2000; Knight et al., Platelets 15: 409-418, 2004; Leong et al., Cytokine16: 106-119, 2001; and Yang et al., Protein Eng. 16: 761-770, 2003). Inone embodiment, antigenic epitopes can be identified and altered bymutagenesis. Methods of identifying antigenic epitopes are known in theart (see for example, Sette et al., Biologicals 29:271-276), as aremethods of mutating such antigenic epitopes.

Methods of Preparing MPPs

MPPs according to the present invention can be prepared by many standardmethods, as known in the art. Modifications to the MPP can be made, forexample, by engineering the nucleic acid encoding the MPP usingrecombinant DNA technology. Alternatively, modifications to the MPP maybe made by modifying the MPP polypeptide itself, using chemicalmodifications and/or limited proteolysis. Combinations of these methodsmay also be used to prepare the MPPs according to the present invention,as is also known in the art.

Preparation of MPPs Using Recombinant Methods

As is known in the art, genetic engineering of a protein generallyrequires that the nucleic acid encoding the protein first be isolatedand cloned. Sequences for various nPPs are available from GenBank asnoted herein. Isolation and cloning of the nucleic acid sequenceencoding these proteins can thus be achieved using standard techniques[see, for example, Ausubel et al., Current Protocols in MolecularBiology, Wiley & Sons, NY (1997 and updates); Sambrook et al., MolecularCloning: A Laboratory Manual, Cold-Spring Harbor Press, NY (2001)]. Forexample, the nucleic acid sequence can be obtained directly from asuitable organism, such as Aeromonas hydrophila, by extracting the mRNAby standard techniques and then synthesizing cDNA from the mRNA template(for example, by RT-PCR). Alternatively, the nucleic acid sequenceencoding the nPP can be obtained from an appropriate cDNA or genomic DNAlibrary by standard procedures. The isolated cDNA or genomic DNA is theninserted into a suitable vector. One skilled in the art will appreciatethat the precise vector used is not critical to the instant invention.Examples of suitable vectors include, but are not limited to, plasmids,phagemids, cosmids, bacteriophage, baculoviruses, retroviruses or DNAviruses. The vector may be a cloning vector or it may be an expressionvector.

Once the nucleic acid sequence encoding the nPP has been obtained,mutations in one or more of the binding domain or activation sequencecan be introduced at specific, pre-selected locations by in vitrosite-directed mutagenesis techniques well-known in the art. Mutationscan be introduced by deletion, insertion, substitution, inversion, or acombination thereof, of one or more of the appropriate nucleotidesmaking up the coding sequence. This can be achieved, for example, by PCRbased techniques for which primers are designed that incorporate one ormore nucleotide mismatches, insertions or deletions. The presence of themutation can be verified by a number of standard techniques, for exampleby restriction analysis or by DNA sequencing.

If desired, after introduction of the appropriate mutation or mutations,the nucleic acid sequence encoding the MPP can be inserted into asuitable expression vector. Examples of suitable expression vectorsinclude, but are not limited to, plasmids, phagemids, cosmids,bacteriophages, baculoviruses and retroviruses, and DNA viruses.

One skilled in the art will understand that the expression vector mayfurther include regulatory elements, such as transcriptional elements,required for efficient transcription of the MPP-encoding sequences.Examples of regulatory elements that can be incorporated into the vectorinclude, but are not limited to, promoters, enhancers, terminators, andpolyadenylation signals. The present invention, therefore, providesvectors comprising a regulatory element operatively linked to a nucleicacid sequence encoding a genetically engineered MPP. One skilled in theart will appreciate that selection of suitable regulatory elements isdependent on the host cell chosen for expression of the geneticallyengineered MPP and that such regulatory elements may be derived from avariety of sources, including bacterial, fungal, viral, mammalian orinsect genes.

For example, a prostate-specific promoter responsive to testosterone andother androgens, can be used to promote gene expression in prostatecells. Examples include, but are not limited to the probasin promoter;the prostate specific antigen (PSA) promoter; the prostate specificmembrane antigen (PSMA) promoter; and the human glandular kallikrein 2(HK2) promoter.

In the context of the present invention, the expression vector mayadditionally contain heterologous nucleic acid sequences that facilitatethe purification of the expressed MPP. Examples of such heterologousnucleic acid sequences include, but are not limited to, affinity tagssuch as metal-affinity tags, histidine tags, avidin/strepavidin encodingsequences, glutathione-S-transferase (GST) encoding sequences and biotinencoding sequences. The amino acids corresponding to expression of thenucleic acids can be removed from the expressed MPP prior to useaccording to methods known in the art. Alternatively, the amino acidscorresponding to expression of heterologous nucleic acid sequences canbe retained on the MPP, provided that they do not interfere with theability of the MPP to target and kill prostate cells.

In one embodiment of the invention, the MPP is expressed as a histidinetagged protein. In another embodiment, the histidine tag is located atthe carboxyl terminus of the MPP.

The expression vectors can be introduced into a suitable host cell ortissue by one of a variety of methods known in the art. Such methods canbe found generally described in Ausubel et al., Current Protocols inMolecular Biology, Wiley & Sons, NY (1997 and updates); Sambrook et al.,Molecular Cloning: A Laboratory Manual, Cold-Spring Harbor Press, NY(2001) and include, for example, stable or transient transfection,lipofection, electroporation, and infection with recombinant viralvectors. One skilled in the art will understand that selection of theappropriate host cell for expression of the MPP will be dependent uponthe vector chosen. Examples of host cells include, but are not limitedto, bacterial, yeast, insect, plant and mammalian cells.

In addition, a host cell may be chosen which modulates the expression ofthe inserted sequences, or modifies and processes the gene product in aspecific, desired fashion. Such modifications (e.g., glycosylation) andprocessing (e.g., cleavage) of protein products may be important for thefunction of the protein. Different host cells have characteristic andspecific mechanisms for the post-translational processing andmodification of proteins and gene products. Appropriate cell lines orhost systems can be chosen to ensure the correct modification andprocessing of the foreign protein expressed. To this end, eukaryotichost cells that possess the cellular machinery for proper processing ofthe primary transcript, and for post-translational modifications such asglycosylation and phosphorylation of the gene product can be used. Suchmammalian host cells include, but are not limited to, CHO, VERO, BHK,HeLa, COS, MDCK, 293, 3T3, WI38.

Methods of cloning and expressing proteins are well-known in the art,detailed descriptions of techniques and systems for the expression ofrecombinant proteins can be found, for example, in Current Protocols inProtein Science (Coligan, J. E., et al., Wiley & Sons, New York). Thoseskilled in the field of molecular biology will understand that a widevariety of expression systems can be used to provide the recombinantprotein. The precise host cell used is not critical to the invention.Accordingly, the present invention contemplates that the MPPs can beproduced in a prokaryotic host (e.g., E. coli, A. salmonicida or B.subtilis) or in a eukaryotic host (e.g., Saccharomyces or Pichia;mammalian cells, e.g., COS, NIH 3T3, CHO, BHK, 293, or HeLa cells; orinsect cells).

The MPPs can be purified from the host cells by standard techniquesknown in the art. If desired, the changes in amino acid sequenceengineered into the protein can be determined by standard peptidesequencing techniques using either the intact protein or proteolyticfragments thereof.

As an alternative to a directed approach to introducing mutations intonaturally occurring pore-forming proteins, a cloned gene expressing apore-forming protein can be subjected to random mutagenesis bytechniques known in the art. Subsequent expression and screening of themutant forms of the protein thus generated would allow theidentification and isolation of MPPs according to the present invention.

The MPPs according to the present invention can also be prepared asfragments or fusion proteins. A fusion protein is one which includes anMPP linked to other amino acid sequences that do not inhibit the abilityof the MPP to selectively target and kill normal prostate cells. In oneembodiment, the other amino acid sequences are no more than 5, 6, 7, 8,9, 10, 20, 30, or 50 amino acid residues in length.

Methods for making fusion proteins are well known to those skilled inthe art. For example U.S. Pat. No. 6,057,133 discloses methods formaking fusion molecules composed of human interleukin-3 (hIL-3) variantor mutant proteins functionally joined to a second colony stimulatingfactor, cytokine, lymphokine, interleukin, hematopoietic growth factoror IL-3 variant. U.S. Pat. No. 6,072,041 to Davis et al. discloses thegeneration of fusion proteins comprising a single chain Fv moleculedirected against a transcytotic receptor covalently linked to atherapeutic protein.

Similar methods can be used to generate fusion proteins comprising MPPs(or variants, fragments, etc. thereof) linked to other amino acidsequences, such as a prostate specific targeting domain (for exampleLHRH or an antibody). Linker regions can be used to space the twoportions of the protein from each other and to provide flexibilitybetween them. The linker region is generally a polypeptide of between 1and 500 amino acids in length, for example less than 30 amino acids inlength. In general, the linker joining the two molecules can be designedto (1) allow the two molecules to fold and act independently of eachother, (2) not have a propensity for developing an ordered secondarystructure which could interfere with the functional domains of the twoproteins, (3) have minimal hydrophobic or charged characteristic whichcould interact with the functional protein domains and/or (4) providesteric separation of the two regions. Typically surface amino acids inflexible protein regions include Gly, Asn and Ser. Other neutral aminoacids, such as Thr and Ala, can also be used in the linker sequence.Additional amino acids can be included in the linker to provide uniquerestriction sites in the linker sequence to facilitate construction ofthe fusions. Other moieties can also be included, as desired. These caninclude a binding region, such as avidin or an epitope, or a tag such asa polyhistidine tag, which can be useful for purification and processingof the fusion protein. In addition, detectable markers can be attachedto the fusion protein, so that the traffic of the fusion protein througha body or cell can be monitored conveniently. Such markers includeradionuclides, enzymes, fluorophores, and the like.

Fusing of the nucleic acid sequences of the MPP with the nucleic acidsequence of another protein (or variant, fragment etc. thereof), can beaccomplished by the use of intermediate vectors. Alternatively, one genecan be cloned directly into a vector containing the other gene. Linkersand adapters can be used for joining the nucleic acid sequences, as wellas replacing lost sequences, where a restriction site was internal tothe region of interest. Genetic material (DNA) encoding one polypeptide,peptide linker, and the other polypeptide is inserted into a suitableexpression vector which is used to transform prokaryotic or eukaryoticcells, for example bacteria, yeast, insect cells or mammalian cells. Thetransformed organism is grown and the protein isolated by standardtechniques, for example by using a detectable marker such asnickel-chelate affinity chromatography, if a polyhistidine tag is used.The resulting product is therefore a new protein, a fusion protein,which has the MPP joined to a second protein, optionally via a linker.To confirm that the fusion protein is expressed, the purified proteincan be, for example, subjected to electrophoresis in SDS-polyacrylamidegels, and transferred onto nitrocellulose membrane filters usingestablished methods. The protein products can be identified by Westernblot analysis using antibodies directed against the individualcomponents, i.e., polyhistidine tag and/or the MPP.

If the MPPs according to the present invention are produced byexpression of a fused gene, a peptide bond serves as the linker betweenthe MPP and the prostate-specific targeting domain. For example, arecombinant fusion protein of a single chain Fv fragment of an antibodyand a pore-forming protein toxin can be made according to methods knownin the art, e.g., Huston et al., Meth. Enzymol. 203:46-88, 1991.

One of ordinary skill in the art will appreciate that the DNA can bealtered in numerous ways without affecting the biological activity ofthe encoded protein. For example, PCR can be used to produce variationsin the DNA sequence which encodes an MPP. Such variations in the DNAsequence encoding an MPP can be used to optimize for codon preference ina host cell used to express the protein, or may contain other sequencechanges that facilitate expression.

Other Methods of Preparing MPPs

The prostate-specific targeting domains and optional linkers noted abovemay be added to the MPPs of the present invention via a covalent ornon-covalent bond, or both. Non-covalent interactions can be ionic,hydrophobic, or hydrophilic, such as interactions involved in aleucine-zipper or antibody-Protein G interaction (Derrick et al., Nature359:752, 1992). Examples of additional non-covalent interactions includebut are not restricted to the following binding pairs: antigen or haptenwith antibody; antibody with anti-antibody; receptor with ligand; enzymeor enzyme fragment with substrate, substrate analogue or ligand; biotinor lectin with avidin or streptavidin; lectin with carbohydrate; pairsof leucine zipper motifs (see, for example, U.S. Pat. No. 5,643,731), aswell as various homodimers and heterodimers known in the art. As isknown in the art, the MPP may be modified to include one member of thebinding pair, and the prostate-specific targeting domain may be modifiedto include the other member of the binding pair.

A covalent linkage may take the form of a disulfide bond. The DNAencoding one of the components can be engineered to contain a uniquecysteine codon. Alternatively, use can be made of a naturally occurringcysteine residue. The second component can be derivatized with asulfhydryl group reactive with the cysteine of the first component.Alternatively, a sulfhydryl group, either by itself or as part of acysteine residue, can be introduced using solid phase polypeptidetechniques. For example, the introduction of sulfhydryl groups intopeptides is described by Hiskey (Peptides 3:137, 1981).

Proteins can be chemically modified by standard techniques to add asulfhydryl group. For example, Traut's reagent (2-iminothiolane-HCl)(Pierce Chemicals, Rockford, Ill.) can be used to introduce a sulfhydrylgroup on primary amines, such as lysine residues or N-terminal amines. Aprotein or peptide modified with Traut's reagent can then react with aprotein or peptide which has been modified with reagents such asN-succinimidyl 3-(2-pyridyldithio) propionate (SPDP) or succinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) (Pierce Chemicals,Rockford, Ill.).

Once the correct sulfhydryl groups are present on each component, thetwo components are purified, sulfur groups on each component arereduced; the components are mixed; and disulfide bond formation isallowed to proceed to completion at room temperature. To improve theefficiency of the coupling reaction, the cysteine residue of one of thecomponents, e.g., cysteine-MPP, can be activated prior to addition tothe reaction mixture with 5,5′-dithiobis(2-nitrobenzoic) acid (DTNB) or2,2′-dithiopyridine, using methods known in the art. Following thereaction, the mixture is dialyzed against phosphate buffered saline toremove unconjugated molecules. Sephadex chromatography or the like isthen carried out to separate the compound of the invention from itsconstituent parts on the basis of size.

The components can also be joined using the polymer,monomethoxy-polyethylene glycol (mPEG), as described in Maiti et al.,Int. J. Cancer Suppl. 3:17-22, 1988.

The prostate-specific targeting domain and the nPP or MPP can also beconjugated through the use of standard conjugation chemistries as isknown in the art, such as, carbodiimide-mediated coupling (for example,DCC, EDC or activated EDC), and the use of 2-iminothiolane to convertepsilon amino groups to thiols for crosslinking andm-maleimidobenzoyl-n-hydroxysuccinimidyl ester (MBS) as a crosslinkingagent. Various other methods of conjugation known in the art can beemployed to join the prostate-specific targeting domain and the nPP orMPP.

Large Scale Preparation of MPPs

The preparation of the MPPs can also be conducted on a large scale, forexample for manufacturing purposes, using standard techniques known inthe art, such as large scale fermentation processes for production ofrecombinant proteins, and ultrafiltration, ion exchange chromatography,immobilized metal ion affinity chromatography for purification ofrecombinant proteins.

Methods of Testing MPPs

The MPPs according to the present invention retain their pore-formingactivity and selectively kill prostate cells. The ability of the MPPs toselectively kill prostate cells can be tested using standard techniquesknown in the art. Exemplary methods of testing candidate MPPs areprovided below and in the Examples included herein. One skilled in theart will understand that other methods of testing candidate MPPs areknown in the art and are also suitable for testing the MPPs according tothe present invention.

In Vitro Methods

MPPs according to the present invention that contain a prostate-specificactivation sequence can be tested for their ability to be cleaved by theappropriate prostate-specific protease according to methods known in theart. For example, the MPP can be incubated with varying concentrationsof the appropriate protease and the incubation products can beelectrophoresed on SDS-PAGE gels and cleavage of the MPP can be assessedby examining the size of the polypeptide on the gel.

In order to determine if the MPPs that have been incubated with proteaseretain pore-forming activity, and thus the ability to kill cells afterincubation with the protease, the reaction products can be tested in ahemolysis assay as is known in the art. An example of a suitable assayis described in Howard, S. P., and Buckley, J. T. 1985. Activation ofthe hole-forming toxin aerolysin by extracellular processing. J.Bacteriol. 163:336-340.

MPPs according to the present invention can be tested for their abilityto kill prostate cells as is known in the art. For example, the abilityof the MPPs to kill prostate cells can be assayed in vitro using asuitable prostate cell line. In general, cells of the selected test cellline are grown to an appropriate density and the candidate MPP is added.After an appropriate incubation time (for example, about 48 to 72hours), cell survival is assessed. Methods of determining cell survivalare well known in the art and include, but are not limited to, theresazurin reduction test (see Fields & Lancaster (1993) Am. Biotechnol.Lab. 11:48-50; O'Brien et al., (2000) Eur. J. Biochem. 267:5421-5426 andU.S. Pat. No. 5,501,959), the sulforhodamine assay (Rubinstein et al.,(1990) J. Natl. Cancer Inst. 82:113-118) or the neutral red dye test(Kitano et al., (1991) Euro. J. Clin. Investg. 21:53-58; West et al.,(1992) J. Investigative Derm. 99:95-100) or trypan blue assay. Numerouscommercially available kits may also be used, for example the CellTiter96® AQueous One Solution Cell Proliferation Assay (Promega).Cytotoxicity is determined by comparison of cell survival in the treatedculture with cell survival in one or more control cultures, for example,untreated cultures and/or cultures pre-treated with a control compound(typically a known therapeutic), or other appropriate control. MPPsconsidered to be effective in killing normal prostate cells are capableof decreasing cell survival, for example, by at least 10%, at least 20%,at least 30%, at least 40%, or at least 50%.

MPPs comprising a prostate-specific targeting domain can be assessed fortheir ability to selectively target prostate cells, for example, bycomparing the ability of the MPP to kill normal prostate cells to itsability to kill cells from other tissues. Alternatively, flow cytometricmethods, as is known in the art, may be used to determine if an MPPcomprising prostate-specific targeting domain is able to selectivelytarget prostate cells. As yet another alternative, the binding ligandfor the prostate-specific targeting domain can be incorporated intoartificial lipid membranes and the ability of the MPP to form channelscan be measured using methods familiar to those skilled in the art.

Assays which can be used to test the MPPs according to the presentinvention for their ability to specifically lyse prostate cells aredescribed for example, in Examples 2 and 3. For example, an MPP having aPSA cleavage site can be assessed for its ability to specifically lysePSA-producing cells compared to its ability to lyse non-PSA producingcells. MPPs according to the present invention when contacted with aPSA-producing cell (such as a prostate cell), promote lysis and death ofthe cell, at lower concentrations than are required to kill a non-PSAproducing cell, for example, by at least 2-fold, 5-fold, 10-fold or100-fold lower concentrations.

A variety of prostate cell-lines suitable for testing the candidate MPPsare known in the art and many are commercially available (for example,from the American Type Culture Collection, Manassas, Va.). Examples ofsuitable prostate cell-lines for in vitro testing include, but are notlimited to PNT1A, PNT2, BPH-1, DuK50, NRP152, PS-1 cell lines.

If necessary, the toxicity of the MPPs to non-prostate cells can also beinitially assessed in vitro using standard techniques. For example,human primary fibroblasts can be transfected in vitro with the MPP andthen tested at different time points following treatment for theirviability using a standard viability assay, such as the assays describedabove, or the trypan-blue exclusion assay. Cells can also be assayed fortheir ability to synthesize DNA, for example, using a thymidineincorporation assay, and for changes in cell cycle dynamics, forexample, using a standard cell sorting assay in conjunction with afluorocytometer cell sorter (FACS).

The activity of MPPs according to the present invention in plasma orserum can also be tested as known in the art. For example, the MPPs canbe incubated with serum for a suitable period of time, after which thedegree of activation of the MPP is measured using, for example,electrophoresis and densitometric analysis of electrophoresed bandscorresponding to activated MPP.

In Vivo Methods

The toxicity of the MPPs according to the present invention can betested in vivo according to methods known in the art. For example, theoverall systemic toxicity of the MPPs can be tested by determining thedose that kills 100% of mice (i.e. LD₁₀₀) following a single intravenousinjection as described in Example 4. Toxicity due to systemic orintraprostatic administration of an MPP can also be assessed in vivo,for example, by administering the MPP to dogs, rats or monkeys.

The ability of the MPPs according to the present invention to decreasethe size of the prostate, thus indicating suitability for the treatmentof BPH can be tested in vivo using animal models known in the art. For,example, the in vivo activity of MPPs can be tested using dogs, ornon-human primates such as the cynomologous monkey, chimpanzee andbaboon. The MPPs can be administered, for example, by perianalintraprostatic injection. Changes in prostate volume afteradministration can be evaluated, for example, by magnetic resonanceimaging or by postmortem examination of the prostate tissue and/ordetermination of prostate weight.

As noted above, MPPs capable of decreasing the size of the prostategland in an animal model, or attenuating further growth of the prostategland are considered to be suitable for the treatment of BPH. Decreasingthe size of the prostate gland refers to a decrease in the weight orvolume of a prostate gland, and attenuating of further growth of theprostate gland refers to the situation where there is minimal or noincrease in the weight or volume of a prostate gland in an animalsubsequent to administration of the test compound. In one embodiment,MPPs contemplated by the present invention, when administered to ananimal, are capable of decreasing prostate gland size, for example, byat least 10%, 20%, 30%, 40%, or 50%.

Determination and Reduction of MPP Antigenicity

Therapeutic proteins may elicit some level of antibody response whenadminstered to a subject, which in some cases can lead to potentiallyserious side effects. Therefore, if necessary, the antigenicity of theMPPs can be assessed as known in the art and described below. Inaddition, methods to reduce potential antigenicity are described.

The kinetics and magnitude of the antibody response to the MPPsdescribed herein can be determined, for example, in immunocompetentmice, and can be used to facilitate the development of a dosing regimenthat can be used in an immunocompetent human. Immunocompetent mice suchas the strain C57-BL6 are administered intravenous doses of the MPP.Mice are sacrificed at varying intervals (e.g. following single dose,following multiple doses) and serum obtained. An ELISA-based assay canbe used to detect the presence of anti-MPP antibodies.

To decrease antigenicity of MPPs according to the present invention, thenative binding domain of the MPP can be functionally deleted andreplaced, for example with a prostate-specific targeting domain asdescribed above. The antigenicity of such MPPs can be determinedfollowing exposure to varying schedules of the MPP which lack portionsof the native binding domain using the methods described above. Anothermethod that can be used to allow continued treatment with MPPs is to usesequentially administered alternative MPPs derived from other nPPs withnon-overlapping antigenicity. For example, an MPP derived fromproaerolysin can be used alternately with an MPP derived fromClostridium septicum alpha toxin or Bacillus thuringiensis delta-toxin.All of these MPPs would target prostate cells, but would not berecognized or neutralized by the same antibodies. Another example is touse an MPP derived from human tissues, such as human perforin producedby cytolytic human T cells. Such MPPs, can be administered and notproduce an antibody response because the proteins are of human origin.

Pharmaceutical Compositions

The present invention provides for pharmaceutical compositionscomprising an MPP and one or more non-toxic pharmaceutically acceptablecarriers, diluents, excipients and/or adjuvants. If desired, otheractive ingredients may be included in the compositions. As indicatedabove, such compositions are used in the treatment of BPH.

The pharmaceutical compositions may comprise from about 1% to about 95%of a MPP of the invention. Compositions formulated for administration ina single dose form may comprise, for example, about 20% to about 90% ofthe MPPs of the invention, whereas compositions that are not in a singledose form may comprise, for example, from about 5% to about 20% of theMPPs of the invention. Concentration of the MPP in the final formulationcan be as low as 0.01 μg/mL. For example, the concentration in the finalformulation can be between about 0.01 μg/mL and about 1,000 μg/mL. Inone embodiment, the concentration in the final formulation is betweenabout 0.01 μg/mL and about 100 μg/mL. Non-limiting examples of unit doseforms include dragées, tablets, ampoules, vials, suppositories andcapsules. Non-limiting examples of unit dose forms include dragées,tablets, ampoules, vials, suppositories and capsules.

The composition can be a liquid solution, suspension, emulsion, tablet,pill, capsule, sustained release formulation, or powder. For solidcompositions (e.g., powder, pill, tablet, or capsule forms),conventional non-toxic solid carriers can include, for example,pharmaceutical grades of mannitol, lactose, starch, sodium saccharine,cellulose, magnesium carbonate, or magnesium stearate. The compositioncan be formulated as a suppository, with traditional binders andcarriers such as triglycerides.

For administration to an animal, the pharmaceutical compositions can beformulated for administration by a variety of routes. For example, thecompositions can be formulated for oral, topical, rectal or parenteraladministration or for administration by inhalation or spray. The termparenteral as used herein includes subcutaneous injections, intravenous,intramuscular, intrathecal, intrasternal injection or infusiontechniques. Direct injection or infusion into the prostate gland is alsocontemplated. Convection enhanced delivery, a standard administrationtechnique for protein toxins, is also contemplated by the presentinvention.

The MPPs can be delivered along with a pharmaceutically acceptablevehicle. Ideally, such a vehicle would enhance the stability and/ordelivery properties. Thus, the present invention also provides forformulation of the MPP with a suitable vehicle, such as an artificialmembrane vesicle (including a liposome, noisome, nanosome and the like),microparticle or microcapsule, or as a colloidal formulation thatcomprises a pharmaceutically acceptable polymer. The use of suchvehicles/polymers may be beneficial in achieving sustained release ofthe MPPs. Alternatively, or in addition, the MPP formulations caninclude additives to stabilise the protein in vivo, such as human serumalbumin, or other stabilisers for protein therapeutics known in the art.

Pharmaceutical compositions for oral use can be formulated, for example,as tablets, troches, lozenges, aqueous or oily suspensions, dispersiblepowders or granules, emulsion hard or soft capsules, or syrups orelixirs. Such compositions can be prepared according to standard methodsknown to the art for the manufacture of pharmaceutical compositions andmay contain one or more agents selected from the group of sweeteningagents, flavoring agents, colouring agents and preserving agents inorder to provide pharmaceutically elegant and palatable preparations.Tablets contain the active ingredient in admixture with suitablenon-toxic pharmaceutically acceptable excipients including, for example,inert diluents, such as calcium carbonate, sodium carbonate, lactose,calcium phosphate or sodium phosphate; granulating and disintegratingagents, such as corn starch, or alginic acid; binding agents, such asstarch, gelatine or acacia, and lubricating agents, such as magnesiumstearate, stearic acid or talc. The tablets can be uncoated, or they maybe coated by known techniques in order to delay disintegration andabsorption in the gastrointestinal tract and thereby provide a sustainedaction over a longer period. For example, a time delay material such asglyceryl monostearate or glyceryl distearate may be employed.

Pharmaceutical compositions for oral use can also be presented as hardgelatine capsules wherein the active ingredient is mixed with an inertsolid diluent, for example, calcium carbonate, calcium phosphate orkaolin, or as soft gelatine capsules wherein the active ingredient ismixed with water or an oil medium such as peanut oil, liquid paraffin orolive oil.

Pharmaceutical compositions formulated as aqueous suspensions containthe active compound(s) in admixture with one or more suitableexcipients, for example, with suspending agents, such as sodiumcarboxymethylcellulose, methyl cellulose, hydropropylmethylcellulose,sodium alginate, polyvinylpyrrolidone, hydroxypropyl-β-cyclodextrin, gumtragacanth and gum acacia; dispersing or wetting agents such as anaturally-occurring phosphatide, for example, lecithin, or condensationproducts of an alkylene oxide with fatty acids, for example,polyoxyethyene stearate, or condensation products of ethylene oxide withlong chain aliphatic alcohols, for example,hepta-decaethyleneoxycetanol, or condensation products of ethylene oxidewith partial esters derived from fatty acids and a hexitol for example,polyoxyethylene sorbitol monooleate, or condensation products ofethylene oxide with partial esters derived from fatty acids and hexitolanhydrides, for example, polyethylene sorbitan monooleate. The aqueoussuspensions may also contain one or more preservatives, for exampleethyl, or n-propyl p-hydroxy-benzoate, one or more colouring agents, oneor more flavoring agents or one or more sweetening agents, such assucrose or saccharin.

Pharmaceutical compositions can be formulated as oily suspensions bysuspending the active compound(s) in a vegetable oil, for example,arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oilsuch as liquid paraffin. The oily suspensions may contain a thickeningagent, for example, beeswax, hard paraffin or cetyl alcohol. Sweeteningagents such as those set forth above, and/or flavoring agents may beadded to provide palatable oral preparations. These compositions can bepreserved by the addition of an anti-oxidant such as ascorbic acid.

The pharmaceutical compositions can be formulated as a dispersiblepowder or granules, which can subsequently be used to prepare an aqueoussuspension by the addition of water. Such dispersible powders orgranules provide the active ingredient in admixture with one or moredispersing or wetting agents, suspending agents and/or preservatives.Suitable dispersing or wetting agents and suspending agents areexemplified by those already mentioned above. Additional excipients, forexample, sweetening, flavoring and coloring agents, can also be includedin these compositions.

Pharmaceutical compositions of the invention can also be formulated asoil-in-water emulsions. The oil phase can be a vegetable oil, forexample, olive oil or arachis oil, or a mineral oil, for example, liquidparaffin, or it may be a mixture of these oils. Suitable emulsifyingagents for inclusion in these compositions include naturally-occurringgums, for example, gum acacia or gum tragacanth; naturally-occurringphosphatides, for example, soy bean, lecithin; or esters or partialesters derived from fatty acids and hexitol, anhydrides, for example,sorbitan monoleate, and condensation products of the said partial esterswith ethylene oxide, for example, polyoxyethylene sorbitan monoleate.The emulsions can also optionally contain sweetening and flavoringagents.

Pharmaceutical compositions can be formulated as a syrup or elixir bycombining the active ingredient(s) with one or more sweetening agents,for example glycerol, propylene glycol, sorbitol or sucrose. Suchformulations can also optionally contain one or more demulcents,preservatives, flavoring agents and/or coloring agents.

The pharmaceutical compositions can be formulated as a sterileinjectable aqueous or oleaginous suspension according to methods knownin the art and using suitable one or more dispersing or wetting agentsand/or suspending agents, such as those mentioned above. The sterileinjectable preparation can be a sterile injectable solution orsuspension in a non-toxic parentally acceptable diluent or solvent, forexample, as a solution in 1,3-butanediol. Acceptable vehicles andsolvents that can be employed include, but are not limited to, water,Ringer's solution, lactated Ringer's solution and isotonic sodiumchloride solution. Other examples include, sterile, fixed oils, whichare conventionally employed as a solvent or suspending medium, and avariety of bland fixed oils including, for example, synthetic mono- ordiglycerides. Fatty acids such as oleic acid can also be used in thepreparation of injectables.

Other pharmaceutical compositions and methods of preparingpharmaceutical compositions are known in the art and are described, forexample, in “Remington: The Science and Practice of Pharmacy” (formerly“Remingtons Pharmaceutical Sciences”); Gennaro, A., Lippincott, Williams& Wilkins, Philadelphia, Pa. (2000).

The pharmaceutical compositions of the present invention described aboveinclude one or more MPPs of the invention in an amount effective toachieve the intended purpose. Thus the term “therapeutically effectivedose” refers to the amount of the MPP that ameliorates the symptoms orcharacteristics of BPH. Determination of a therapeutically effectivedose of a compound is well within the capability of those skilled in theart. For example, the therapeutically effective dose can be estimatedinitially either in cell culture assays, or in animal models, such asthose described herein. Animal models can also be used to determine theappropriate concentration range and route of administration. Suchinformation can then be used to determine useful doses and routes foradministration in other animals, including humans, using standardmethods known in those of ordinary skill in the art.

Therapeutic efficacy and toxicity can also be determined by standardpharmaceutical procedures such as, for example, by determination of themedian effective dose, or ED₅₀ (i.e. the dose therapeutically effectivein 50% of the population) and the median lethal dose, or LD₅₀ (i.e. thedose lethal to 50% of the population). The dose ratio betweentherapeutic and toxic effects is known as the “therapeutic index,” whichcan be expressed as the ratio, LD₅₀/ED₅₀. The data obtained from cellculture assays and animal studies can be used to formulate a range ofdosage for human or animal use. The dosage contained in suchcompositions is usually within a range of concentrations that includethe ED₅₀ and demonstrate little or no toxicity. The dosage varies withinthis range depending upon the dosage form employed, sensitivity of thesubject, and the route of administration and the like.

The exact dosage to be administered to a subject can be determined bythe practitioner, in light of factors related to the subject requiringtreatment. Dosage and administration are adjusted to provide sufficientlevels of the MPP and/or to maintain the desired effect. Factors whichmay be taken into account when determining an appropriate dosage includethe severity of the disease state, general health of the subject, age,weight, and gender of the subject, diet, time and frequency ofadministration, drug combination(s), reaction sensitivities, andtolerance/response to therapy. Dosing regimens can be designed by thepractitioner depending on the above factors as well as factors such asthe half-life and clearance rate of the particular formulation.

Pharmaceutically effective amounts MPPs of the present invention can beformulated with pharmaceutically acceptable carriers for parenteral,oral, nasal, rectal, topical, transdermal administration or the like,according to conventional methods. Formulations may further include oneor more diluents, fillers, emulsifiers, preservatives, buffers,excipients, and the like, and may be provided in such forms as liquids,powders, emulsions, suppositories, liposomes, transdermal patches andtablets, for example. Slow or extended-release delivery systems,including any of a number of biopolymers (biological-based systems),systems employing liposomes, and polymeric delivery systems, can also beutilized with the compositions described herein to provide a continuousor long-term source of MPP. Such slow release systems are applicable toformulations, for example, for oral, topical and parenteral use. Theterm “pharmaceutically acceptable carrier” refers to a carrier mediumwhich does not interfere with the effectiveness of the biologicalactivity of the active ingredients and which is not toxic to the host orpatient. One skilled in the art may formulate the compounds of thepresent invention in an appropriate manner, and in accordance withaccepted practices, such as those disclosed in Remington: The Scienceand Practice of Pharmacy, Gennaro, ed., Mack Publishing Co., Easton Pa.,19th ed., 1995

In one embodiment, the MPP is conjugated to a water-soluble polymer,e.g., to increase stability or circulating half life or reduceimmunogenicity. Clinically acceptable, water-soluble polymers include,but are not limited to, polyethylene glycol (PEG), polyethylene glycolpropionaldehyde, carboxymethylcellulose, dextran, polyvinyl alcohol(PVA), polyvinylpyrrolidone (PVP), polypropylene glycol homopolymers(PPG), polyoxyethylated polyols (POG) (e.g., glycerol) and otherpolyoxyethylated polyols, polyoxyethylated sorbitol, or polyoxyethylatedglucose, and other carbohydrate polymers. Methods for conjugatingpolypeptides to water-soluble polymers such as PEG are described, e.g.,in U.S. patent Pub. No. 20050106148 and references cited therein.

Use of MPPs for Treatment of Benign Prostatic Hyperplasia (BPH)

The MPPs according to the present invention selectively kill normalprostate cells relative to cells from other normal tissues. Thus, theMPPs according to the present invention are useful in the treatment orprevention of BPH.

In one embodiment, treatment of BPH refers to a decrease in the size ofthe prostate gland, in a subject with BPH. The size of the prostategland can be measured in terms of its volume, by methods known in theart including, for example, planimetry, prolate ellipse volumecalculation (HWL), and an ellipsoid volume measurement technique.Prostate size can also be measured directly, for example by digitalrectal examination, or rectal ultrasound or cytoscopy, or indirectly,for example, by measuring changes in the levels of blood PSA or changesin the proportions of free and total PSA in the blood.

In one embodiment, administration of MPP decreases the volume of theprostate gland in a subject. For example, the disclosed methods canreduce prostate volume, for example, by at least 10%, by at least 20%,or by at least 30% by at least 40%, or by at least 50%.

In another embodiment, treatment of BPH refers to the decrease in thedegree of severity of one or more symptoms of BPH. Symptoms of BPHinclude changes or problems with urination, such as a hesitant,interrupted or weak stream, urgency and leaking or dribbling, or morefrequent urination, especially at night. These symptoms are also knownas lower urinary tract symptoms (LUTS). LUTS can be measured as known inthe art using the American Urological Association (AUA) Symptom Index,the Madsen-Iversen Scoring System, or the Boyarsky System.

In another embodiment, treatment of BPH refers to the prevention orinhibition of continued growth of the prostate gland and can be measuredby a reduction in the rate of increase in the volume or the rate ofincrease of blood PSA or reduction in symptoms of BPH as describedabove.

Combination Therapy

The MPPs according to the present invention can be used alone or incombination with one or more additional treatments for BPH. Theadditional treatments for BPH include administration of drugs such asα-1-adrenoreceptor antagonists and 5-α reductase inhibitors,phytotherapies, surgical procedures, and minimally invasive techniques.

Examples of α-1-adrenoreceptor antagonists are alfuzosin/prazosin,tamsulosin, terazosin, and doxazosin. Examples of 5-α reductaseinhibitors are finasteride and dutasteride.

Examples of phytotherapies include Saw palmetto berry/dwarf palm(Serenoa repens), African plum bark (Pygeum africanum), South Africanstar grass/beta-sitosterol (Hipoxis rooperi), Purple cone flower(Echinacea purpurea), Pumpkin seeds (Cucurbita pepo), Rye (Secalecereale), and Stinging nettle (Urtica dioica).

Examples of surgical procedures are transurethral resection of theprostate (TURP), transurethral needle ablation (TUNA), transurethralincision of the prostate (TUIP), transurethral microwave thermotherapy(TUMT), laser prostatectomy, balloon dilation, electrical vaporizationand open prostatectomy.

If necessary to reduce a systemic immune response to the MPPs,immunosuppressive therapies can be administered in combination with theMPPs. Examples of immunosuppressive therapies include, but are notlimited to, systemic or topical corticosteroids (Suga et al., Ann.Thorac. Surg. 73:1092-7, 2002), cyclosporin A (Fang et al., Hum. GeneTher. 6:1039-44, 1995), cyclophosphamide (Smith et al., Gene Ther.3:496-502, 1996), deoxyspergualin (Kaplan et al., Hum. Gene Ther.8:1095-1104, 1997) and antibodies to T and/or B cells [e.g. anti-CD40ligand, anti CD4 antibodies, anti-CD20 antibody (Rituximab)] (Manning etal., Hum. Gene Ther. 9:477-85, 1998). Such agents can be administeredbefore, during, or subsequent to administration of the MPP. The MPPs ofthe present invention may be administered separately, sequentially orsimultaneously with the above noted treatments.

Administration of MPPs

A therapeutically effective amount of an MPP according to the presentinvention, or a nucleic acid encoding an MPP, can be administeredlocally or systemically using methods known in the art, to subjectshaving BPH.

In one embodiment, the MPPs are injected into the prostate gland(intraprostatically) in a subject having BPH. For example, anadministration approach similar to the multiple injection approach ofbrachytherapy can be used, in which multiple aliquots of the purifiedpeptides, adapted as compositions or formulations and in the appropriatedosage form, may be injected using a needle through the perineum.

In addition, or alternatively, the MPPs can be administeredsystemically, for example intravenously, intramuscularly,subcutaneously, or orally, to a subject having BPH.

A therapeutically effective amount of an MPP refers to an amountsufficient to achieve a desired biological effect, for example an amountthat is effective to decrease the size (i.e. volume and/or weight) ofthe prostate gland, or attenuate further growth of the prostate gland,or decrease symptoms of BPH. In one embodiment, it is an amountsufficient to decrease the signs or symptoms of BPH in a subject. Inparticular examples, it is an amount effective to decrease the volume ofa prostate gland by at least 10%, 20%, 30%, 40%, or 50%. In anotherembodiment, it is an amount sufficient to prevent further increase involume or weight of the prostate gland. Effective doses can beextrapolated from dose-response curves derived from in vitro or animalmodel test systems.

An effective amount of an MPP can be administered in a single dose, orin several doses, for example daily, during a course of treatment.However, the effective amount of MPP will be dependent on the subjectbeing treated, the severity and type of the condition being treated, andthe manner of administration. In one embodiment, a therapeuticallyeffective amount of an MPP can vary from about 0.01 to 50 μg per gramprostate weight, administered intraprostatically. In another embodiment,a therapeutically effective amount of an MPP can vary from about 0.02 to40 μg per gram prostate weight, administered intraprostatically. Inanother embodiment, a therapeutically effective amount of an MPP canvary from about 0.02 to 35 μg per gram prostate weight, administeredintraprostatically. In another embodiment, a therapeutically effectiveamount of an MPP can vary from about 0.03 to 25 μg per gram prostateweight, administered intraprostatically. In another embodiment, atherapeutically effective amount of an MPP can vary from about 0.04 to20 μg per gram prostate weight, administered intraprostatically. Inanother embodiment, a therapeutically effective amount of an MPP canvary from about 0.04 to 10 μg per gram prostate weight, administeredintraprostatically.

In one embodiment, an effective intravenous dose intraprostatically anMPP for a 70 kg human is from about 1 mg to about 10 mg of MPP. Inanother embodiment an effective intravenous dose is from about 1 mg toabout 5 mg. In another embodiment, an effective intravenous dose is fromabout 1 mg to about 3 mg. In still another embodiment, an effectiveintravenous dose is about 2.8 mg. In one embodiment, an effectiveintraprostatic dose of an MPP for a 70 kg human is from about 10 mg toabout 100 mg of MPP. In another embodiment, an effective intraprostaticdose of an MPP for a 70 kg human is from about 10 mg to about 50 mg ofMPP. In another embodiment, an effective intraprostatic dose of an MPPfor a 70 kg human is from about 10 mg to about 30 mg of MPP. In anotherembodiment, an effective intraprostatic dose of an MPP is about 28 mgfor a 70 kg human.

In Vivo Expression of MPPs

As an alternative to (or in addition to) administration of MPPs to treatBPH, long term or systemic treatment of BPH can be achieved byexpressing nucleic acids encoding MPPs in vivo.

Nucleic Acids Encoding MPPs

The present invention contemplates the use of nucleic acids or DNAmolecules encoding MPPs for the treatment of BPH. Such DNA molecules canbe obtained through standard molecular biology laboratory techniques andthe sequence information disclosed herein.

Suitable DNA molecules and nucleotide include those which hybridizeunder stringent conditions to the DNA sequences disclosed, or fragmentsthereof, provided that they encode a functional MPP. Hybridizationconditions resulting in particular degrees of stringency vary dependingupon the nature of the hybridization method and the composition andlength of the hybridizing DNA used. Generally, the temperature ofhybridization and the ionic strength (especially the Na⁺ concentration)of the hybridization buffer determines hybridization stringency.Calculations regarding hybridization conditions required for attainingparticular amounts of stringency are discussed by Sambrook et al.(Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, N.Y., 1989,Chapters 9 and 11). Hybridization with a target probe labeled with[³²P]-dCTP is generally carried out in a solution of high ionic strengthsuch as 6.times.SSC at a temperature that is about 5-25° C. below themelting temperature, T_(m). An example of stringent conditions is a saltconcentration of at least about 0.01 to 1.0 M Na ion concentration (orother salts) at pH 7.0 to 8.3 and a temperature of at least about 30° C.for short probes (e.g. 10 to 50 nucleotides). Stringent conditions canalso be achieved with the addition of destabilizing agents such asformamide. For example, conditions of 5.times.SSPE (750 mM NaCl, 50 mMNa phosphate, 5 mM EDTA, pH 7.4) at 25-30° C. are suitable forallele-specific probe hybridizations.

The degeneracy of the genetic code further allows for variations in thenucleotide sequence of a DNA molecule while maintaining the amino acidsequence of the encoded protein. For example, the amino acid Ala isencoded by the nucleotide codon triplet GCT, GCG, GCC and GCA. Thus, thenucleotide sequence could be changed without affecting the amino acidcomposition of the encoded protein or the characteristics of theprotein. Based upon the degeneracy of the genetic code, variant DNAmolecules may be derived from a reference DNA molecule using standardDNA mutagenesis techniques as described above, or by synthesis of DNAsequences. DNA sequences which do not hybridize under stringentconditions to the DNA sequences disclosed by virtue of sequencevariation based on the degeneracy of the genetic code are alsocomprehended by this disclosure.

The present invention provides methods of expressing MPPs, for example amodified proaerolysin polypeptide in a cell or tissue in vivo. In oneexample, transfection of the cell or tissue occurs in vitro. In thisexample, the cell or tissue (such as a graft) is removed from a subjectand then transfected with an expression vector containing a cDNAencoding the protein of interest. The transfected cells will producefunctional protein and can be reintroduced into the subject. In anotherexample, a nucleic acid encoding the protein of interest is administeredto a subject directly (such as intravenous, or intraprostate), andtransfection occurs in vivo.

The scientific and medical procedures required for human celltransfection are now routine. A general strategy for transferring genesinto donor cells is disclosed in U.S. Pat. No. 5,529,774. Generally, agene encoding a protein having therapeutically desired effects is clonedinto a viral expression vector, and that vector is then introduced intothe target organism. The virus infects the cells, and produces theprotein sequence in vivo, where it has its desired therapeutic effect(Zabner et al. Cell 75:207-16, 1993).

It may only be necessary to introduce the DNA or protein elements intocertain cells or tissues, for example, the prostate. However, in someinstances, it may be more therapeutically effective and simple to treatall of a subject's cells, or more broadly disseminate the vector, forexample by intravascular (i.v.) or oral administration.

The nucleic acid sequence encoding the MPP is under the control of asuitable promoter. Suitable promoters which can be used include, but arenot limited to, the gene's native promoter, retroviral LTR promoter, oradenoviral promoters, such as the adenoviral major late promoter; theCMV promoter; the RSV promoter; inducible promoters, such as the MMTVpromoter; the metallothionein promoter; heat shock promoters; thealbumin promoter; the histone promoter; the x-actin promoter; TKpromoters; B19 parvovirus promoters; and the ApoAI promoter. In oneexample, the promoter is a prostate-specific promoter, such as aprobasin promoter. However the disclosure is not limited to specificforeign genes or promoters.

The recombinant nucleic acid can be administered to the subject by knownmethods which allows the recombinant nucleic acid to reach theappropriate cells. These methods include injection, infusion,deposition, implantation, or topical administration. Injections can beintradermal or subcutaneous. The recombinant nucleic acid can bedelivered as part of a viral vector, such as avipox viruses, recombinantvaccinia virus, replication-deficient adenovirus strains or poliovirus,or as a non-infectious form such as naked DNA or liposome encapsulatedDNA, as further described below.

Adenoviral vectors include essentially the complete adenoviral genome(Shenk et al., Curr. Top. Microbiol. Immunol. 111: 1-39, 1984).Alternatively, the adenoviral vector is a modified adenoviral vector inwhich at least a portion of the adenoviral genome has been deleted. Inone example, the vector includes an adenoviral 5′ ITR; an adenoviral 3′ITR; an adenoviral encapsidation signal; a DNA sequence encoding atherapeutic agent; and a promoter for expressing the DNA sequenceencoding a therapeutic agent. The vector is free of at least themajority of adenoviral E1 and E3 DNA sequences, but is not necessarilyfree of all of the E2 and E4 DNA sequences, and DNA sequences encodingadenoviral proteins transcribed by the adenoviral major late promoter.In another example, the vector is an adeno-associated virus (AAV) suchas described in U.S. Pat. No. 4,797,368 (Carter et al.) and inMcLaughlin et al. (J. Virol. 62:1963-73, 1988) and AAV type 4 (Chioriniet al. J. Virol. 71:6823-33, 1997) and AAV type 5 (Chiorini et al. J.Virol. 73:1309-19, 1999).

Such a vector can be constructed according to standard techniques, usinga shuttle plasmid which contains, beginning at the 5′ end, an adenoviral5′ ITR, an adenoviral encapsidation signal, and an E1a enhancersequence; a promoter (which may be an adenoviral promoter or a foreignpromoter); a tripartite leader sequence, a multiple cloning site (whichmay be as herein described); a poly A signal; and a DNA segment whichcorresponds to a segment of the adenoviral genome. The DNA segmentserves as a substrate for homologous recombination with a modified ormutated adenovirus, and may encompass, for example, a segment of theadenovirus 5′ genome no longer than from base 3329 to base 6246. Theplasmid can also include a selectable marker and an origin ofreplication. The origin of replication may be a bacterial origin ofreplication. A desired DNA sequence encoding a therapeutic agent can beinserted into the multiple cloning site of the plasmid.

Examples of vectors which can be used to practice the methods disclosedherein include, but are not limited to, those disclosed in: WO 95/27512to Woo et al.; WO 01/127303 to Walsh et al.; U.S. Pat. No. 6,221,349 toCouto et al.; U.S. Pat. No. 6,093,392 to High et al.

Clinical Trials

One skilled in the art will appreciate that, following the demonstratedeffectiveness of MPPs for the treatment of BPH in in vitro and in animalmodels, the MPPs should be tested in clinical trials in order to furtherevaluate their efficacy in the treatment of BPH and to obtain regulatoryapproval for therapeutic use. As is known in the art, clinical trialsprogress through phases of testing, which are identified as Phases I,II, III, and IV.

Initially the MPPs will be evaluated in a Phase I trial. Typically PhaseI trials are used to determine the best mode of administration (forexample, by pill or by injection), the frequency of administration, andthe toxicity for the compounds. Phase I studies frequently includelaboratory tests, such as blood tests and biopsies, to evaluate theeffects of the potential therapeutic in the body of the patient. For aPhase I trial, a small group of patients with BPH are treated with aspecific dose of MPP. During the trial, the dose is typically increasedgroup by group in order to determine the maximum tolerated dose (MTD)and the dose-limiting toxicities (DLT) associated with the compound.This process determines an appropriate dose to use in a subsequent PhaseII trial.

A Phase II trial can be conducted to further evaluate the effectivenessand safety of the MPP. In Phase II trials, the MPP is administered togroups of patients with BPH, using the dosage found to be effective inPhase I trials.

Phase III trials focus on determining how the MPP compares to thestandard, or most widely accepted, treatment. In Phase III trials,patients are randomly assigned to one of two or more “arms”. In a trialwith two arms, for example, one arm will receive the standard treatment(control group) and the other arm will receive MPP treatment(investigational group).

Phase IV trials are used to further evaluate the long-term safety andeffectiveness of an MPP. Phase IV trials are less common than Phase I,II and III trials and take place after the MPP has been approved forstandard use.

Eligibility of Patients for Clinical Trials

Participant eligibility criteria can range from general (for example,age, sex, type of disease) to specific (for example, type and number ofprior treatments, disease characteristics, blood cell counts, organfunction). In one embodiment, eligible patients have been diagnosed withBPH. Eligibility criteria may also vary with trial phase. Patientseligible for clinical trials can also be chosen based on objectivemeasurement of urinary obstruction, and failure to respond to oraltreatment for BPH. For example, in Phase I and II trials, the criteriaoften exclude patients who may be at risk from the investigationaltreatment because of abnormal organ function or other factors. In PhaseII and III trials additional criteria are often included regardingdisease type and stage, and number and type of prior treatments.

Phase I trials usually comprise 15 to 30 participants for whom othertreatment options have not been effective. Phase II trials typicallycomprise up to 100 participants who have already received drug therapyor surgery, but for whom the treatment has not been effective.Participation in Phase II trials is often restricted based on theprevious treatment received. Phase III trials usually comprise hundredsto thousands of participants. This large number of participants isnecessary in order to determine whether there are true differencesbetween the effectiveness of MPP and the standard treatment. Phase IIImay comprise patients ranging from those newly diagnosed with BPH tothose with extensive disease in order to cover the disease continuum.

One skilled in the art will appreciate that clinical trials should bedesigned to be as inclusive as possible without making the studypopulation too diverse to determine whether the treatment might be aseffective on a more narrowly defined population. The more diverse thepopulation included in the trial, the more applicable the results couldbe to the general population, particularly in Phase III trials.Selection of appropriate participants in each phase of clinical trial isconsidered to be within the ordinary skills of a worker in the art.

Assessment of Patients Prior to Treatment

Prior to commencement of the study, several measures known in the artcan be used to first classify the patients. Patients can first beassessed, for example, using the benign hyperplasia symptom index foundon the Family Practice Notebook website. Patients can also be classifiedaccording to the type and/or stage of their disease and/or by prostatesize.

Administration of MPP in Clinical Trials

MPP is typically administered to the trial participants by injection. Inone embodiment, the MPP is administered by intraprostatic injection.

A range of doses of the MPP can be tested. Provided with informationfrom preclinical testing, a skilled practitioner could readily determineappropriate dosages of MPP for use in clinical trials. In oneembodiment, a dose range is from about 0.01 μg/g prostate to about 50μg/g prostate. In one embodiment, a dose range is from about 0.02 μg/gprostate to about 40 μg/g prostate. In one embodiment, a dose range isfrom about 0.02 μg/g prostate to about 35 μg/g prostate. In oneembodiment, a dose range is from about 0.03 μg/g prostate to about 25μg/g prostate. In one embodiment, a dose range is from about 0.044 μg/gprostate to about 20 μg/g prostate. In one embodiment, a dose range isfrom about 0.04 μg/g prostate to about 10 μg/g prostate. In oneembodiment, a dose range is from about 0.1 μg/g prostate to about 5 μg/gprostate. In one embodiment, a dose range is from about 0.2 μg/gprostate to about 3 μg/g prostate. In one embodiment, a dose range isfrom about 0.5 μg/g prostate to about 2 μg/g prostate.

Pharmacokinetic Monitoring

To fulfill Phase I criteria, distribution of the MPP is monitored, forexample, by chemical analysis of samples, such as blood or urine,collected at regular intervals. For example, samples can be taken atregular intervals up until about 72 hours after the start of infusion.

If analysis is not conducted immediately, the samples can be placed ondry ice after collection and subsequently transported to a freezer to bestored at −70° C. until analysis can be conducted. Samples can beprepared for analysis using standard techniques known in the art and theamount of MPP present can be determined, for example, byhigh-performance liquid chromatography (HPLC).

Pharmacokinetic data can be generated and analyzed in collaboration withan expert clinical pharmacologist and used to determine, for example,clearance, half-life and maximum plasma concentration.

Monitoring of Patient Outcome

The endpoint of a clinical trial is a measurable outcome that indicatesthe effectiveness of a compound under evaluation. The endpoint isestablished prior to the commencement of the trial and will varydepending on the type and phase of the clinical trial. Examples ofendpoints include, for example decline in prostate volume, decline inblood PSA levels, improved urinary tract symptoms, improved urinaryflow, and reduction in acute urinary retention. Other endpoints includetoxicity and quality of life.

Pharmaceutical Kits

The present invention additionally provides for therapeutic kits orpacks containing one or more MPPs or a pharmaceutical compositioncomprising one or more MPPs for use in the treatment of BPH. The MPPscan be provided in the kit in unit dosage form. Individual components ofthe kit can be packaged in separate containers, associated with which,when applicable, can be a notice in the form prescribed by agovernmental agency regulating the manufacture, use or sale ofpharmaceuticals or biological products, which notice reflects approvalby the agency of manufacture, use or sale for human or animaladministration. The kit can optionally further contain one or more othertherapeutic agents for use in combination with the MPPs of theinvention. The kit may optionally contain instructions or directionsoutlining the method of use or dosing regimen for the MPPs and/oradditional therapeutic agents.

When the components of the kit are provided in one or more liquidsolutions, the liquid solution can be an aqueous solution, for example asterile aqueous solution. In this case the container means may itself bean inhalant, syringe, pipette, eye dropper, or other such likeapparatus, from which the composition may be administered to a patientor applied to and mixed with the other components of the kit.

The components of the kit may also be provided in dried or lyophilisedform and the kit can additionally contain a suitable solvent forreconstitution of the lyophilised components. Irrespective of the numberor type of containers, the kits of the invention also may comprise aninstrument for assisting with the administration of the composition to apatient. Such an instrument may be an inhalant, syringe, pipette,forceps, measured spoon, eye dropper or similar medically approveddelivery vehicle.

The invention will now be described with reference to specific examples.It will be understood that the following examples are intended todescribe embodiments of the invention and are not intended to limit theinvention in any way.

EXAMPLES Example 1 Generation of MPPS Activated by PSA

This example describes methods used to produce the MPPs according toembodiments of the invention as shown in Table 6, which are activated byPSA. These MPPs are derived from proaerolysin. One skilled in the artwill understand that similar methods can be used to produce other MPPswhich are activated by PSA or any other prostate-specific protease. Suchproteins can be produced by substituting the furin sequence ofproaerolysin with a prostate-specific protease cleavage site, such as aPSA-specific cleavage sequence.

TABLE 6 Comparison of MPPs with an activation sequence containing aprotease cleavage site cleaved by PSA with wild-type ProaerolysinComparison to wt Proaerolysin MPP Change(s) madeADSKVRRARSVDGAGQGLRLEIPLD (SEQ ID NO.) (SEQ ID NO.) (aa 424-448 of SEQID NO: 2) MPP1 KVRRAR (aa 427-432 of ADSHSSKLQSVDGAGQGLRLEIPLD) (3 & 4)SEQ ID NO: 2) changed to (aa 424-448 of SEQ ID NO: 4) HSSKLQ (5) MPP2KVRRARSV (aa 427-434 of ADSHSSKLQSADGAGQGRLEIPLD (6 & 7) SEQ ID NO: 2)changed to (aa 424-448 of SEQ ID NO: 7) HSSKLQSA (8) MPP3 KVRRAR (aa427-432 of ADSQFYSSNSVDGAGQGLRLEIPLD (9 & 10) (SEQ ID NO: 2) changed to(aa 424-448 of SEQ ID NO: 10) QFYSSN (11) MPP4 KVRRAR (aa 427-432 ofADSGISSFQSSVDGAGQGLRLEIPLD (12 & 13) (SEQ ID NO: 2) changed to (aa424-448 of SEQ ID NO: 13) GISSFQS (14)

The MPPs shown in Table 6 include a proaerolysin sequence (wild-type PAshown in SEQ ID NOS: 1 and 2) in which the six amino acid furin proteaserecognition site (amino acids 427-432 of SEQ ID NO: 2) was replaced witha PSA cleavage site. For example, MPP1 (SEQ ID NOS: 3 and 4), includes aproaerolysin sequence in which the furin cleavage site was replaced bythe PSA substrate HSSKLQ (SEQ ID NO: 5).

Recombinant PCR was used to substitute the furin site of aerolysin(amino acids 427-432 of SEQ ID NO: 2) with a PSA-specific cleavage site(SEQ ID NO: 5, 8, 11 or 14) using methods previously described (Valletteet al., Nucl. Acids Res. 17:723-33, 1988). Briefly, recombinant PCR wasperformed in a final volume of 50 μl which contained 0.2 mMdeoxynucleoside triphosphate (dNTPs), 0.5 μM forward and reverseprimers, 0.1 μg template DNA and 2.5 units cloned pfu polymerase in pfuReaction Buffer [20 mM Tris-HCl (pH 8.8), 10 mM KCl, 10 mM (NH₄)₂SO₄, 2mM MgSO₄, 0.1% Triton X-100, and 0.1 mg/ml BSA].

Screening transformed cells for the proaerolysin insert was performed byPCR using Taq polymerase. A cocktail was prepared in PCR reaction buffer[50 mM KCl, 1.5 mM MgCl₂, and 10 mM Tris-HCl (pH 9.0)] containing 0.2 mMdNTPs, 0.5 μM forward and reverse primers and 5 units of Taq polymerase.Ten μl samples of this cocktail were aliquoted into 0.2 ml tubes andtransformed cells were added using sterile toothpicks.

The final PCR products were digested using appropriate restrictionenzymes, then ligated into the cloning vector pTZ18u (BioRad) foramplification. Briefly, restriction digests were performed at 37° C. for90 minutes in Pharmacia One-Phor-All buffer [10 mM Tris-acetate (pH7.5), 10 mM Mg-acetate, and 50 mM K-acetate] containing about one unitof restriction enzyme for every μg of DNA. The resulting insert, andpTZ18u vector DNA, were mixed together in a ratio of approximately 5:1and heated at 45° C. for 15 minutes. Subsequently, the samples werediluted in One-Phor All buffer and ATP added to a final concentration of1 mM for cohesive-end ligations or 0.5 mM for blunt-end ligations. Then,11 units of T4 DNA ligase were added to each sample and the samplesmixed gently. Ligations were carried out at 13° C. for 4 hours(cohesive-end ligations) or 16 hours (blunt-end ligations).

DNA sequencing was performed to ensure the correct substitutions weremade. The insert was subsequently isolated from the cloning vector andsubcloned into the broad-host-range plasmid pMMB66HE (Furste et al.,Gene 48:119-131, 1986) for expression in E. coli. E. coli DH5α cellswere made competent using the CaCl₂ wash method described previously(Cohen et al. Proc. Nat. Acad. Sci. USA 69:2110-4, 1972). Cells inlog-phase (OD₆₀₀=0.4-0.7) were harvested by centrifugation and washed in¼ volume of cold 100 mM MgCl₂. The cells were pelleted again, andresuspended in two volumes of cold 100 mM CaCl₂. The cells were thenincubated on ice for approximately 45 minutes. The cells were thencentrifuged and resuspended in 1/10 volume of 100 mM CaCl₂. Incubationcontinued for an additional 45 minutes before the addition of glycerolto a final concentration of 15%. Competent cells were stored at −70° C.until use.

Transformation of recombinant plasmids into competent E. coli cells wasperformed according to the method of Inoue et al. (Gene 96: 23-8, 1990).Competent cells (200 μl aliquots) were incubated with 0.5-10 ng of DNAfor one hour on ice. The cells were then subjected to heat shock at 42°C. for 4 minutes. The cells were quickly transferred back onto ice for 5minutes. Subsequently, 500 μl of LB media was added to each sample andthe cells incubated for 1 hour at 37° C. with mild agitation. Aliquots(150 μl) were plated onto LB agar containing 50 μg/ml ampicillin. Theseplates were incubated overnight at 37° C.

Recombinant pMMB66HE clones were transferred into Aeromonas salmonicidastrain CB3 (see Buckley, Biochem. Cell. Biol. 68:221-4, 1990) byconjugation using the filter-mating technique of Harayama et al. (Mol.Gen. Genet. 180:47-56, 1980). Use of this protease-deficient strain ofA. salmonicida resulted in production of MPPs that were not contaminatedby activated aerolysin and resulted in production of large quantities ofprotein. The MPPs were purified by hydroxyapatite chromatography and ionexchange chromatography as previously described (Buckley, Biochem. Cell.Biol. 68:221-4, 1990). This method resulted in preparations of the MPPsidentical from batch to batch.

Example 2 MPP1 Specifically LYSES PSA-Producing Cells In Vitro

This example describes methods used to determine the specificity of theMPPs according to embodiments of the invention as described inExample 1. Such methods can be used to test the specificity of MPPs thatinclude a PSA-specific cleavage site.

MPP1 was tested against PSA-producing LNCaP cells (American Type CultureCollection, Manassas, Va.) and non-PSA-producing TSU cells (Dr. T.Itzumi, Teikyo University, Japan). Cells were incubated in the presenceof 10⁻¹² M to 10⁻⁶ M MPP1 for 24 hours. Subsequently, cells were countedand scored for percent viable cells based on ability to exclude TrypanBlue. Concentration required to kill 50% of cells (IC₅₀) was determinedfor MPP1 against both LNCaP and TSU lines.

The LD₅₀ for MPP1 against PSA-producing cells was 10⁻¹⁰ M. In contrast,against non-PSA producing TSU cells the LD₅₀ was about 5×10⁻⁸ M. Thisresult demonstrates that MPP1 is specifically activated by PSA asevidenced by a 500-fold difference in toxicity against PSA-producingversus non-PSA producing human cell lines.

Example 3 MPP1 is not Activated in Blood Containing PSA

MPPs which include a PSA cleavage site should not be activated in blood,because PSA is enzymatically inactivated in the blood due to thepresence of a large molar excess of serum protease inhibitors such asalpha-1-antichymotrypsin and alpha-2-macroglobulin.

To test for non-specific activation of MPP1 by other serum proteases andPSA in human serum, a sensitive hemolysis assay was performed asfollows. Red blood cells (RBCs, 2% v/v) were added to plasma or buffercontaining MPP1±PSA. The extent of hemolysis was assayed by measuringrelease of hemoglobin into the supernatant. Addition of 0.1% Tritonresults in 100% hemolysis within a few seconds and was used as thepositive control. Amount of hydrolysis was expressed as a ratio ofsample absorbance at 540 nm to absorbance of Triton treated sample.Pre-incubation of the MPP1 (10⁻⁸ M) with PSA in aqueous buffer alone for1 hour prior to adding RBCs resulted in about 45% hemolysis (FIG. 2).

To determine whether MPP1 becomes activated in human plasma, MPP1 (10⁻⁸M) was incubated in 50% human plasma for 1 hour. In a relatedexperiment, excess PSA (10,000 ng/ml) was first added to the humanplasma and allowed to incubate for several hours. MPP1 containing plasma±PSA was then incubated with human RBCs (2% v/v). The addition of MPP1to human plasma, or human plasma spiked with high concentration of PSA,resulted in no appreciable hemolysis (i.e. <1% of Triton control, FIG.2). These results demonstrate that MPP1 can be administered systemicallywithout any significant activation in the blood, even if the bloodcontains measurable PSA.

Example 4 In Vitro and In Vivo Toxicity of MPP1, MPP2, and MPP3

This example describes methods used to determine the in vitro and invivo toxicity of MPPs.

To determine in vitro toxicity, a cell viability assay was performed asfollows. EL4 mouse T-cell lymphoma cells (ATCC TIB-39) were cultured at10⁵ cells per well in MTS/PMS Cell Titer 96 (Promega). MPP1, MPP2 andMPP3 at 1×10⁻¹³ M−1×10⁻⁷ M were added as shown in FIG. 3, and incubatedwith the cells for 4 hours at 37° C. Cell viability was subsequentlydetermined by reading the plate on a plate reader, as directed by themanufacturer of the MTS/PMS kit. As shown in FIG. 3, the MPPs are lesstoxic than wild-type proaerolysin, with an LC₅₀ of 4×10⁻⁹ (MPP1), 1×10⁻⁹(MPP2), and 1×10⁻⁷ (MPP3), in contrast to an LC₅₀ of 1.5×10⁻¹° forwild-type.

To determine in vivo toxicity, MPPs were administered to miceintravenously. Wild-type proaerolysin (SEQ ID NO: 2) was highly toxic tomice; a dose of 1 μg caused death within one hour and the LD₁₀₀ at 24hours (i.e. the dose that kills 100% of animals within 24 hours)following a single IV injection was 0.1 μg. In contrast, the LD₁₀₀ ofMPP1 (SEQ ID NO: 4) at 24 hours post injection was 25-fold higher (i.e.2.5 μg total dose).

Example 5 Preparation of MPP5 (A Histidine-Tagged MPP)

A histidine-tagged MPP according to the present invention (MPP5) wasprepared as follows. The plasmid insert containing the gene encodingMPP1 was modified to improve ease of purification and yield. A stretchof 35 nucleotides, including the ribosome binding site and the ATG startcodon, that when transcribed, forms a secondary loop structure, whichcauses reduction in production of the protein (Burr et al., J.Bacteriol. 183: 5956-63, 2001) was modified to prevent loop formation,leaving 23 nucleotides upstream of the ATG start codon (Diep et al.,Mol. Microbiol. 30: 341-52, 1998). This increased the amount of MPP thatcould be released into the culture supernatant of CB3 (Burr et al., J.Bacteriol. 183: 5956-63, 2001). The construct with the new upstreamsequence is designated as the γ123 promoter construct. The change wascarried out by digesting the MPP1 construct with KpnI and EcoRI,followed by ligation of the resulting fragment into the KpnI/EcoRI siteof γ123-aerA::pTZ18U. The resulting construct was calledγ123-MPP1::pTZ18U.

Addition of a His tag was accomplished using a 2-step QuikChange(Stratagene) protocol. In the first step, 3 His residues were added tothe end of the γ123-MPP1 DNA by synthesizing two primers (the 3 CATcodons encode the extra His residues):

EndHis1 (sense): (SEQ ID NO: 26) 5′-GCT GCC AAT CAA CAT CAT CAT TAA CGGCAG CGC-3′ EndHis1 (antisense) (SEQ ID 27) 5′-GCG CTG CCG TTA ATG ATGATG TTG ATT GGC AGC-3′

These primers were used in the protocol suggested by Stratagene for theQuikChange kit.

Once the addition of the DNA for the 3 His residues was confirmed bysequencing, a second QuikChange reaction was performed to addnucleotides for the final 3 His residues. In this step, the primers usedwere:

EndHis2 (sense) (SEQ ID NO: 28) 5′-GCC AAT CAA CAT CAT CAT CAT CAT CATTAA CGG CAG CGC-3′ EndHis2 (antisense) (SEQ ID NO: 29) 5′-GCC AAT CAACAT CAT CAT CAT CAT CAT TAA CGG CAG CGC-3′

After the second round of PCR, clones were screened and sequenced toconfirm that the 6 His residues were correctly inserted at the end ofγ123-MPP1 in the correct reading frame, and that no other changes weremade to the γ123-MPP1 sequence. The resulting plasmid was namedγ123-MPP5::pTZ18U, and the γ123-PSAH6 insert region was sequenced (seeFIG. 34).

The nucleic acid sequence of γ123-MPP5 differs from that of the MPP1construct in the region before the ATG start codon, with the γ123promoter replacing the normal aerA upstream sequence. The sequence alsohas the additional CAT repeats immediately before the TAA stop codon.When the open reading frame of γ123-MPP5 was translated into the aminoacid sequence, the only difference seen compared to the MPP1 amino acidsequence was the addition of 6 histidine residues at the C-terminus ofthe protein.

Cloning of γ123-MPP5 into pMMB208 Expression Vector

The γ123-MPP5 created as a result of the addition of γ123 promoter andHis-tag, was cloned into plasmid pMMB208. This plasmid was chosen as itconfers chloramphenicol resistance, it contains an IPTG inducible tacpromoter, and it can be mobilized into Aeromonas salmonicida bytransconjugation. Thus the γ123-PSA PAH6 insert was cloned into theHindIII and EcoRI sites of the pMMB208 plasmid. The resultant plasmid,γ123-MPP5::208, was transconjugated into A. salmonicida strain CB3 forthe production of MPP5.

In order to purify GLP MPP5, 30 L of sterile defined media set at aninitial pH of 6.90±0.15 and temperature 27° C. was inoculated withapproximately 1% v/v of shake flask inoculum. The pH of the medium wasmaintained at 6.90±0.15 by automatic addition of sterile acid/alkaliduring the fermentation. Fermenter RPM and SLM were adjusted to maintainDissolved Oxygen Tension (DOT) of ≧20% in the vessel at all times.Expression of MPP5 was induced by addition of isopropylthiogalactopyranoside (IPTG) when the cell density reached an OD at 600nm of 0.3-0.6. Expressed protein was secreted into the medium andharvested several hours after induction. The supernatant containing MPP5was recovered using a 60 SP CUNO filter. Following cell separation, thesupernatant was concentrated using a 30,000 NMWC TFF membrane.Concentrated supernatant was purified using two chromatographic stepsthat yielded protein of acceptable purity; nickel chelatingchromatography, followed by anion exchange chromatography. The MPP5containing fractions from the anion exchange chromatography step werepooled and the resulting material was concentrated to approximately 1mg/mL by ultrafiltration and subsequently diafiltered into formulationbuffer. Purified MPP5 was sterile filtered using a 0.22 μm absolutefilter in a certified biological safety cabinet and frozen to −70° C.until required. Final yield of purified MPP5 using this process wasapproximately 100 mg/L.

Example 6 An Acute Intraprostatic or Intravenous Bolus InjectionToxicity Study of MPP5 in the Albino Rat

This example shows preliminary results of a study indicating thatintraprostatic administration of a histidine-tagged MPP (MPP5) to ratswas able to decrease the weight of the prostate gland.

Summary of Results

The objective of this study was to confirm the maximum tolerated dose(MTD) and to investigate the potential toxicity and toxicokinetics of anMPP (MPP5) following a single intraprostatic injection or intravenousbolus injection to male rats followed by a 1, 14, or 28-day observationperiod. MPP5 is a histidine tagged proaerolysin molecule which comprisesa PSA cleavage site.

The study design is detailed in Tables 7 and 8:

TABLE 7 Study Design MTD Study Dose Volume Number of Dose Level (μL)Males Group Treatment (μg) IP IV IP IV 6 MPP5 10 20 500 3 3 7 MPP5 20 20500 3 3 8 MPP5 40 20 500 3 3 9 MPP5 50 — 500 — 3 IP—Intraprostatic,IV—Intravenous, Animals were terminated following a 48-hour observationperiod

TABLE 8 Main and Toxicokinetic Study Design Dose Main Study Terminal TKLevel Sacrifice Males per Group Study Group Treatment (μg) Route Day 2Day 15 Day 29 Animals 1 Control 0 IP 7 7 6 9 2 MPP5 2 IP 7 7 6 9 3 MPP510 IP 7 7 6 9 4 MPP5 25 IP 7 7 6 9 5 MPP5 25 IV 7 7 6 9IP—Intraprostatic (20 μL), IV—Intravenous (0.05 mL), TK—Toxicokinetic

The following were evaluated: clinical signs, body weight, foodconsumption, opthalmology, hematology, serum chemistry, urinalysis,toxicokinetics, macroscopic observations at necropsy, organ weights andhistopathology. There were no effects on body weight, body weight gain,food consumption, opthalmology or urinalysis parameters. One MTD animaltreated intraprostatically at 40 μg was found dead on Day 2. Prior todeath there were no treatment-related clinical signs. Dark foci wereseen in the stomach and a dark area and swelling were noted at theinjection site (prostate). Two main study animals treatedintraprostatically at 25 μg were found dead on Day 2. Prior to deaththere were no treatment-related clinical signs noted for one animal, butfur staining (muzzle), blue skin, decreased activity, weakness,decreased muscle tone, clear liquid discharge (periorbital), pale eyes,labored breathing, lying on side and cold to touch were noted in theother animal.

Macroscopic findings in one animal included: a dark area at theinjection site (prostate); dark foci in the testes; pale, clear fluid inthe abdomen cavity, including pale material adjacent to the liver. Inthe other animal, lesions were seen in the fat and jejunum, includingdiscoloration. Adhesions were seen in the liver, thickening of thepancreas, multiple dark areas in the stomach and thymus, and dark fociin the abdominal fat, adjacent to the epididymides. A specific cause ofdeath was not identified for these two males, however, it was assumedthat the severity of the prostatic inflammation with its proximity tokidneys and concurrent systemic degenerative alterations contributed tothe death of these animals.

Treatment-related clinical signs were seen in animals treated at all MTDIP dose levels. Red fur staining was seen in some animals from allgroups at one or more sites including the muzzle, jaw, forepaw,periorbital, and ventral/dorsal cervical between Days 1 and 4. Blue skindiscoloration (surgical site, abdominal, urogenital or inguinal sites)was noted at 20 and 40 μg in ⅓ animals each, including urogenitalswelling in one animal at 40 μg. In MTD animals treated intravenously,red fur staining (muzzle and/or periorbital), were noted with a greaterincidence in animals treated at 50 μg.

Red fur staining (muzzle) was predominantly seen in main study animalsfrom all IP dose levels including the controls, but generally with ahigher incidence in treated animals. This clinical sign was consideredlikely treatment-related. Red fur staining (muzzle) was also observed inall main study IV animals. Additional clinical signs were noted at thesite of injection (tail) and included skin scabs and/or redness or otherdiscoloration. In addition, two animals required tail amputation due tolesions and suspected self-mutilation. These latter changes suggested anirritant potential of the test article formulation which was furthersupported following histopathological examinations.

A dose-related increase was seen in some white cell parameters in mainstudy IP animals on Day 2. WBC, neutrophil, monocyte and basophil countswere increased in all treated groups at 10 and/or 25 μg. Increases werealso seen in MCHC at 25 μg and in MPV at all dose levels.

Decreases were recorded in percent and absolute reticulocytes at alldose levels. A slight, but dose-related increase in activated partialthromboplastin time was noted at all IP dose levels. Similar changeswere observed in animals treated intravenously at 25 μg. WBC, neutrophiland basophil counts were increased. MCHC, MPV and red cell distributionwidth (RDW) were also increased and percent and absolute reticulocyteswere decreased. In animals terminated after 14 days of observation,increases were seen in RDW and MPV only and after 28 days, there were nodifferences noted in hematology parameters. Changes noted in animalsterminated after 1 day of observation were considered to be testarticle-related. Results shown following the 15-day and 28-dayobservation period demonstrate recovery from these changes.

Dose related increases in mean aspartate aminotransferase and alanineaminotransferase concentration were seen on Day 2 and considered markedat 25 μg. Increases in direct bilirubin, urea, creatinine andtriglyceride concentration were also noted at 25 μg IP. Decreases wereseen in glucose concentration at 25 μg and albumin concentration wasdecreased at ≧10 μg with an associated decrease in albumin to globulinratio and decrease in total protein concentration at 25 μg only. Inanimals treated 25 μg IV, a slight increase was seen in urea and calciumconcentration and a slight increase in globulin concentration was alsonoted with a corresponding decrease in albumin to globulin ratio. Thesechanges were considered related to the inflammation observed at the siteof injection. On Day 29, an increase in alanine aminophosphataseconcentration and a decrease in indirect bilirubin concentration werenoted at 25 μg IP only.

The composite terminal half-life was estimated at 12.8 hours forintravenous administration. The C_(max) could be back extrapolated totime 0 hours with a value of 81.3 ng/mL. The observed peak value (at thefirst sampling time) was 74.3 ng/mL. The systemic clearance (CL) andvolume of distribution (V_(z)) was estimated as 46.3 mL/h and 841 mL,respectively. Following intraprostatic administration, observed t_(max)occurred at 4 hours post dose for all cases, with peak levels of 2.95and 3.51 ng/mL at 10 and 25 μg, respectively. The observed AUC_(0-tlast)appeared to decrease at the higher dose, due to the different t_(last)observed. Dose linearity for the intraprostatic route was assessed usingC_(max) and AUC_(0-tlast). Both dose normalized exposure parameters weredecreased from 10 μg to 25 μg and the corresponding estimatedbioavailabilities were 23.7 and 4.38%, all of which was indicative oflimited absorption of MPP5 into the systemic circulation from theprostate with increasing dose level.

During the MTD phase, single IP doses of MPP5 at 40 μg in male rats wereassociated with mortality with no specific cause of death. A single IVdose of MPP5 in male rats was tolerated up to a maximum dose of 50 μg.

During the main study phase, single IP injection of MPP5 in male ratsresulted in the death of 2 rats at 25 μg, prostate injection sitemacroscopic, microscopic and organ weight alterations at ≧2 μg. Thecause of death of the two males could not be ascribed with certainty butit was assumed that the severity of the prostatic inflammation andassociated changes contributed to the deterioration/death of theseanimals. The other test article related changes were observed at 2, 15,and/or 29 days of the recovery period. Macroscopic, microscopic andorgan weight alterations in other tissues were considered to besecondary to the prostate injection site changes.

Single IV injection of MPP5 in male rats resulted in tail vein injectionsite macroscopic and/or microscopic alterations at a dose of ≧25 μg.These changes were observed at 2, 15 and 29 days of the recovery periodwith progressive recovery at 25 μg and were indicative of an irritanteffect of the test article. Macroscopic and microscopic alterations inother tissues were considered to be secondary to the injection sitechanges. Liver and spleen organ weight changes on Day 2 were withoutmicroscopic correlation and recovered by Day 15.

In conclusion, the administration of MPP5 by single intraprostaticinjection at dose levels up to 40 μg or intravenous injection at doselevels up to 50 μg resulted in mortality at 25 μg and 40 μg IP with noclear cause of death, however the extent of the test article relatedprostatic inflammation and with proximity to the kidneys and concurrentsystemic degeneration were considered contributory factors to thedeaths. Mostly reversible changes were seen in clinical signs (≧2 μg),hematology and clinical biochemistry parameters at ≧10 μg. Pathologicalchanges persisted at all dose levels in a dose-related fashion butshowed evidence of regression in animals treated intravenously.Consequently, the no-observable-effect-level (NOEL) was not determinedfor either the intraprostatic or the intravenous route.

Experimental Procedures 3.1. Test System

A total of 180 male Sprague Dawley (Crl:CD® (SD) IGS BR) rats (Rattusnorvegicus) were used. At the start of treatment, animals were 12 to 15weeks of age and ranged in weight from 399 to 495 g.

3.2 Veterinary Treatments

On Days 4 and 7, tail amputations were performed on animals 5010 and5004, respectively, due to suspected self-mutilation. As these animalswere treated intravenously, the amputated tissue was retained in neutralbuffered 10% formalin for pathological evaluation.

Prior to treatment, all animals were weighed and randomly assigned totreatment groups using a computer-based randomization procedure.Randomization was by stratification using body weight as the parameter.Animals in poor health were not assigned to groups. The study design isdetailed in Table 9 (MTD) and Table 7 (Main).

TABLE 9 MTD Study Dose Dose Volume Level (μL) Number of Males GroupTreatment (μg) IP IV IP IV 6 MPP5 10 20 500 3 3 7 MPP5 20 20 500 3 3 8MPP5 40 20 500 3 3 9 MPP5 50 — 500 — 5 IP—Intraprostatic, IV—Intravenous

Animals assigned to the MTD Study were terminated following a 48-hourobservation period. On Day 1, animals 6001 and 8001 assigned to the IPdose regimen were replaced by spare animals 6101 and 8101, respectively,due to a technical error (overdosed by 10 times their assigned dose).Later the same day, animal 8001 (400 μg) was found dead approximately 4hours post dose and animal 6001 (100 μg) was euthanized at the end ofDay 1 due to poor condition. These animals were subject to necropsyincluding a detailed external and internal examination, however; notissues were retained for histopathological examination. Prior to death,clinical signs for animal 6001 included weakness, decreased muscle tone,eyes partly closed, cold to touch, decreased activity and abnormal gait.Clinical signs for animal 8001 included lying on side, laboredbreathing, decreased respiratory rate, blue skin, pale eyes, cold totouch, decreased activity, weakness and decreased muscle tone. Grossexamination for animal 6001 revealed only a single dark area at theadministration site (prostate). For animal 8001, bilateral darkdiscoloration was noted in the mandibular lymph nodes and swelling wasnoted at the injection site (prostate). These deaths were consideredprobably related to the high MPP5 administration and are reported inorder to provide further reference for the MTD in this study.

On Day 13 (MTD study), additional animals were added at a dose level of50 μg via the intravenous route in order to further explore MPP5toxicity via this route due to limited observations seen at ≦40 μg.

Prior to initiation of dosing, animal 1025 was considered unsuitable foruse in the study due to a malocclusion and was replaced by a spareanimal, which became animal 1125. All animals remaining unassigned togroups were released from the study and their disposition documented.

3.3. Test and Vehicle Control Articles

The test article was MPP5 (Lot Number PTIC-MF-PAL-DS-001), at aconcentration of 3.2 mg/mL. The test article was colorless solid whenfrozen, and was stored at −20° C., out of direct light. The vehiclecontrol was Phosphate Buffered Saline-EDTA, pH 7.4.

3.4 Preparation of Dose Formulations

Dose formulations were prepared on the day of use. On each day,appropriate amounts of the 3.2 mg/mL stock solution of MPP5 was measuredand diluted with appropriate amounts of PBS, 1 mM EDTA.

3.5 Administration of Test/Control Article MTD Study

Groups of three rats were dosed either intraprostatically orintravenously as described in section 3.2 and in Table 9, on a singleday and observed for clinical signs and potential toxicity for up to 48hours post dose. For intravenous administration, the test article wasadministered by intravenous injection, via the tail vein at a dosevolume of 0.5 mL.

For intraprostatic groups, prior to dose administration, animals wereanesthetized using isoflurane. At least 1 hour prior to surgery and upto 2 days following surgery, animals received an intramuscularantibiotic injection of Benzathine penicillin G and Procaine PenicillinG (0.1 mL). Animals also received a subcutaneous injection ofBuprenorphine (0.05 mg/kg) on the day of surgery.

Using a scalpel blade, a midline incision of approximately 2 cm wasmade, starting 0.5 cm cranially of the penis. The abdomen was cut on thesame length. The two ventral lobes of the prostate were localized and 20μL of formulated MPP5 or PBS/EDTA pH 7.4 was injected into the rightventral lobe using an appropriate syringe. Prior to closing, the sitewas irrigated with warm (approximately 37° C.) 0.9% Sodium ChlorideInjection, U.S.P. The site was closed in layers using appropriate suturematerial.

For animals assigned to the intravenous route, the test article wasadministered intravenously, via the tail vein at a dose volume of 0.5mL.

Main Study

Animals were dosed according to the procedures established during theMTD phase. For animals assigned to the intravenous route, the testarticle was administered intravenously, via the tail vein at a dosevolume of 0.05 mL. After completion of treatment, main study animalswere maintained undosed for a 1, 14 or 28-day recovery period.

3.6 Observations

Clinical Observations: All animals were observed twice daily (once onthe day of arrival and necropsy) for mortality and signs of ill healthand/or reaction to treatment throughout the study. In addition, adetailed examination was performed at least once prior to the start oftreatment and daily throughout the treatment and recovery periods (mainand MTD study animals).

Body Weights: Individual body weights were measured for all animals onthe day of randomization and twice weekly throughout treatment andrecovery periods (main study animals). In addition, each mainstudy/recovery animal was weighed (fasted) before scheduled necropsy.MTD study animals were weighed prior to dosing and prior to terminalsacrifice.

Food Consumption: Individual food consumption for all main study animalswas measured weekly commencing the last week of the pretreatment periodand throughout the treatment and recovery periods.

Opthalmology: Once prior to the start of treatment (all animals) andagain prior to necropsy (main study animals), funduscopic (indirectopthalmoscopy) and biomicroscopic (slit lamp) examinations wereperformed by a board-certified veterinary ophthalmologist.

Laboratory Investigations: Blood sampling for hematology and serumchemistry testing was performed on all main study animals at necropsy onDays 2, 15 and 29. Food was removed overnight from animals prior toblood sampling. Blood samples were collected from the abdominal aortaunder isoflurane anesthesia.

Urine samples were collected prior to necropsy on Days 2, 15 and 29 frommain study animals placed in metabolism cages for an approximate 16-hourcollection period, during which the animals were deprived of food.

The hematological parameters examined were: activated partialthromboplastin time, blood cell morphology, erythrocyte indices (MCV,MCH, MCHC and RDW); hematocrit hemoglobin, mean platelet volume plateletcount prothrombin time, red blood cell count, reticulocyte count(absolute and percent), white blood cell count (total, absolute andpercent differential).

The Serum Chemistry, Parameters examined were: A/G ratio (calculated)alanine aminotransferase, albumin, alkaline phosphatase, aspartateaminotransferase, blood urea nitrogen, calcium chloride cholesterolcreatinine, globulin (calculated), glucose, inorganic phosphoruspotassium, sodium, total bilirubin, total protein, triglycerides.

The Urinalysis Parameters examined were: bilirubin, blood color andappearance, creatinine, glucose, ketones, microscopy of centrifugeddeposit nitrite, pH, protein, specific gravity urobilinogen volume.

Immunogenicity Evaluation (Day 14 and Day 28 Main Study Only)

Blood samples were collected from each main study rat pre-dose(baseline) from a jugular vein and at terminal sacrifice from theabdominal aorta following isoflurane anesthesia (along with bloodsampling for clinical pathology). Samples were placed in serumseparation tubes, inverted several times, allowed to clot at roomtemperature for 20 to 30 minutes, and then centrifuged at approximately1200 g for 10 minutes at approximately 4° C.

3.7 Toxicokinetics

On Study Day 1, blood samples (4.5 mL) were collected by venipuncture ofthe jugular vein into K₃ EDTA tubes alternately from three toxicokineticstudy rats per group per time point, at pre-dose, 15, and 30 minutes and1, 2, 4, 8, 24 and 48 hours post-dose. Blood samples were placedimmediately on wet ice until separated by refrigerated centrifugation(approximately 2 to 8° C.) at approximately 2700 rpm for 10 minutes.Plasma was separated, transferred into a second tube and placed on dryice. Plasma samples were stored at approximately −20° C. and analyzedfor levels of MPP5.

Plasma samples were analyzed by Enzyme Linked Immunosorbent Assay(ELISA) based on the antibody sandwich principle. The capture antibody(mouse anti-aerolysin monoclonal antibody) specific to MPP5 was coatedonto the 96-well microtitre plate to create the solid phase, whichcaptured the analyte present in the standards and quality controlsamples. The secondary antibody (rabbit anti-aerolysin polyclonalantibody) that binds to a different epitope of the analyte molecule, wasthen added to complete the antibody-analyte-antibody sandwich. Thedetection antibody enzyme conjugate (goat anti-rabbit IgG, horse radishperoxidase conjugate) that binds to the constant region of the rabbitIgG antibody, was then added. The captured conjugate was visualizedusing the tetramethylbenzidine substrate and measured at 450 nm using aSpectraMAX plate reader.

For the intraprostatic dose route, non-compartmental toxicokineticanalysis was performed on the plasma concentration data. As practical,toxicokinetic analysis included assessment of the t_(max), C_(max), AUC,k and t_(1/2). The t_(max) and C_(max) are observed values. Wherepossible, the AUC parameter was calculated by the trapezoidal rulemethod (Gibaldi and Perrier, 1982) using the standard computer softwareprogram WinNonlin (Version 3.2). The k was determined by linearregression analysis of selected time points in the apparent terminalphase of the concentration vs. time curves. The apparent terminalhalf-life was calculated as follows:

t _(1/2) =In2/k.

For the intravenous dose route, non-compartmental toxicokinetic analysiswas performed on the plasma concentration data. As practical,toxicokinetic analysis included assessment of the t_(max), C_(max), AUC,k, t_(1/2), V_(z) and CL. The C_(max) will be back-extrapolated to time0 hour. The AUC parameter was calculated by the trapezoidal rule method(Gibaldi and Perrier, 1982) using the standard computer software programWinNonlin (Version 3.2). The k was determined by linear regressionanalysis of selected time points in the apparent terminal phase of theconcentration vs. time curves. The apparent terminal half-life wascalculated as follows: t_(1/2)=In2/k. Clearance (CL) was calculated byDose/AUC and the apparent volume of distribution (V_(Z)) was calculatedas CL/k.

3.8. Terminal Procedures

Gross Pathology: MTD study animals found dead during the study weresubject to necropsy without tissue preservation. All main study andrecovery animals found dead during the study were subjected to necropsyand tissue samples were preserved.

On completion of the treatment and recovery period, all survivinganimals were exsanguinated from the abdominal aorta following isofluraneanesthesia and blood sample collection for laboratory investigations. Inorder to avoid autolytic change, a complete gross pathology examinationof the carcass was performed as soon as possible after euthanasia of allmain study animals.

Organ Weights: For each main study animal euthanized at schedulednecropsy, the following organs were dissected free of fat and weighed:adrenal glands, brain, heart, kidneys, liver, lungs, testes, pituitary,prostate, spleen, thymus, thyroid lobes (with parathyroids). Pairedorgans were weighed together and organ weight ratios relative to bodyweights were calculated.

Tissue Preservation: On completion of the necropsy of each main studyanimal, the following tissues and organs were retained: Abnormalities,animal identification, adrenals, aorta (thoracic), bone and marrow(sternum), brain (cerebrum, cerebellum, midbrain and medulla oblongata),cecum, colon, duodenum, epididymis, esophagus, eyes, Harderian glands,heart (including section of aorta), ileum, injection site (prostate)Groups 1 to 4, injection site (tail vein) Group 5, jejunum, kidneys,lacrimal glands, liver (sample of 2 lobes), lungs (sample of 2 lobes),lymph nodes (mandibular and mesenteric), mammary gland (inguinal), nasalcavities and sinuses (3 levels), optic nerves, pancreas, pituitary,prostate (uninjected lobes), rectum, salivary gland, sciatic nerve,seminal vesicles, skeletal muscle, skin (inguinal), spinal cord(cervical), spleen, stomach, testes, thymus, thyroid lobes (andparathyroids), tongue, trachea, ureter (bilateral), urinary bladder.

Neutral buffered 10% formalin was used for tissue fixation andpreservation except for epididymis, eyes, optic nerves and testes, whichwere fixed in Zenker's fluid (euthanized animals only). For alleuthanized animals, 3 femoral bone marrow smears, were prepared andstained. The smears were retained but not evaluated.

Histopathology: Tissues were embedded in paraffin wax, sectioned (nasalcavities and sinuses, sternum and tail vein injection site weredecalcified prior to sectioning), and stained with hematoxylin and eosinand examined histopathologically as follows:

Groups 1, 4 and 5: Tissues listed under tissue preservation (exceptanimal identification and rectum)

Group 2 and Group 3: Tissues showing treatment-related findings, allgross lesions and target tissues listed below:

Tissue samples of target tissues including brain, heart, kidneys, liver,lymph nodes, injection site (prostate), prostate (uninjected lobes),spleen, thyroid lobes (and parathyroids), ureter and urinary bladder,were processed and examined for all main study animals in all dosegroups. Optic nerves, parathyroid glands and mammary gland were onlyexamined histopathologically if present in routine sections of eyes,thyroid and skin, respectively.

Statistical Analyses

Numerical data obtained during the conduct of the study were subjectedto calculation of group means and standard deviations. For eachparameter of interest, group variances were compared using Levene's testat the 0.05 significance level. When differences between group varianceswere not found to be significant, a parametric one-way analysis ofvariance (ANOVA) was performed. If significant differences among themeans were indicated by the ANOVA (p≦0.05), then Dunnett's “t” test wasused to perform the group mean comparisons between the control group andeach treated group.

Whenever Levene's test indicates heterogeneous group variances (p≦0.05),the non-parametric Kruskal-Wallis test was used to compare allconsidered groups. If the Kruskal-Wallis test was significant (p≦0.05),then the significance of the differences between the control group andeach treated group was assessed using Dunn's test.

For each pairwise group comparison of interest, significance wasreported at the 0.05, 0.01 and 0.001 levels.

Results and Discussion 1. Dose Formulation Analysis

The results of pH, osmolality and density assessments of the doseformulations used in the study are indicated in Table 10.

TABLE 10 Dose Formulation pH, Osmolality and Density Dose Group LevelOsmolality Density No. (μg) Route pH (mOsm/kg) (g/cm³) 1 0 IP 7.39 3001.0037 2 2 IP 7.38 299 1.0030 3 10 IP 7.36 298 1.0041 4 25 IP 7.36 2951.0049 5 25 IV 7.36 301 0.9999 6 10 IV/IP 7.35/7.35 308/3040.9941/1.0043 7 20 IV/IP 7.35/7.34 307/302 1.0052/1.0002 8 40 IV/IP7.35/7.32 305/297 1.0048/1.0040 9 50 IV 7.37 308 1.0057

2. Mortality MTD Study

On Day 2, animal 8101 (40 μg, IP) was found dead. Prior to death, therewere no treatment-related clinical signs. At necropsy, dark foci wereseen in the stomach and a dark area and swelling were noted at theinjection site (prostate). No clear cause of death was determined.

Main Study

On Day 2, animals 4005 and 4013 (25 μg, IP) were found dead. Prior todeath, there were no treatment-related clinical signs noted for animal4013. Fur staining (muzzle), blue skin, decreased activity, weakness,decreased muscle tone, clear liquid discharge (periorbital), pale eyes,labored breathing, lying on side and cold to touch were noted for animal4005.

At necropsy for animal 4005, macroscopic findings included: a dark areaat the injection site (prostate); dark foci in the testes; pale, clearfluid in the abdominal cavity, including pale material adjacent to theliver. For animal 4013, lesions were seen in the fat and jejunum,including discoloration. Adhesions were seen in the liver, thickening ofthe pancreas, multiple dark areas in the stomach and thymus, and darkfoci in the abdominal fat, adjacent to the epididymis. The cause ofdeath in both cases was uncertain, however, it was assumed that theseverity of the prostatic inflammation observed histopathologically withits proximity to kidneys and concurrent systemic degenerativealterations contributed to the death of these animals.

3. Clinical Observations MTD Study

Treatment-related effects were seen in animals treated at all MTD IPdose levels (10 to 40 μg). Red fur staining was seen in ⅔ animals fromall groups at one or more sites including the muzzle, jaw, forepaw,periorbital, and ventral/dorsal cervical between Days 1 and 4. Blue skindiscoloration (surgical site, abdominal, urogenital or inguinal sites)was noted at 20 and 40 μg in ⅓ animals each, including urogenitalswelling in animal 8002 (40 μg). Although these observations may berelated to the surgical procedure, the incidence and severity wasincreased at 40 μg.

In animals treated intravenously, treatment-related clinical signs werelimited to red fur staining (muzzle and/or periorbital), which werenoted with a greater incidence in animals treated at 50 μg. Up to 5/5high dose animals were seen with these signs compared to ⅓ that wereobserved with these signs at <50 μg during Days 1 to 4.

Main Study Day 2 Termination

Red fur staining (muzzle, periorbital, and/or cranium) was noted in someanimals from all IP dose levels including 3/7 control animals, 1/7animals at 2 μg, 5/7 animals at 10 μg and 3/7 animals at 25 μg. Animal4014 (25 μg) was observed with reddish discharge from both eyes andanimal 4015 had decreased activity and was cold to touch prior tonecropsy on Day 2.

4. Food Consumption

There was no effect on food consumption.

5. Opthalmology

There were no opthalmologic findings.

6. Hematology

In IP animals terminated on Day 2, a dose-related 2-fold or greaterincrease was seen in some white cell parameters. White blood cell (WBC),neutrophil, monocyte and basophil counts were increased in all treatedgroups, attaining statistical significance at 10 and/or 25 μg.Statistically significant increases were also seen in mean corpuscularhemoglobin concentration (MCHC) at 25 μg and in mean platelet volume(MPV) at all dose levels. Decreases were recorded in percent andabsolute reticulocytes at all dose levels. A slight, but dose-relatedincrease in activated partial thromboplastin time was noted at all IPdose levels, attaining statistical significance at 25 μg.

Similar changes were observed in animals treated intravenously with 25μg MPP5. WBC, neutrophil and basophil counts were increased. MCHC, MPVand red cell distribution width (RDW) were also increased and percentand absolute reticulocytes were decreased. In animals terminated after14 days of observation, increases were seen in RDW and MPV only. After28 days of observation, there were no differences noted in hematologyparameters. The changes noted in animals terminated after 1 day ofobservation were considered test article-related and were correlatedhistopathologically. The results shown following the 15-day and 28-dayobservation period demonstrate evidence of recovery from these changes.Other minor differences were considered incidental and unrelated totreatment.

7. Serum Chemistry

On Day 2, dose related increases, attaining statistical significance at25 μg IP, were seen in mean aspartate aminotransferase and alanineaminotransferase concentration. These were considered marked changes at25 μg. Increases in direct bilirubin, urea, creatinine and triglycerideconcentration were also noted at 25 μg IP. Decreases were seen inglucose concentration at 25 μg. Albumin concentration was decreased at≧10 μg with an associated decrease in albumin to globulin ratio anddecrease in total protein concentration at 25 μg only.

In animals treated 25 μg IV, a slight increase was seen in urea andcalcium concentration. A slight increase in globulin concentration wasalso noted with a corresponding decrease in albumin to globulin (A/G)ratio. The changes in globulin (and consequently in A/G ratio) wereconsidered possibly related to inflammation at the injection site.

At Day 15, statistically significant decreases were seen in triglycerideconcentration at all dose levels including IV treated animals. Thesewere considered of no toxicological significance and possibly related tominor alterations in lipid metabolism.

At Day 29, an increase in alanine aminophosphatase concentration and adecrease in indirect bilirubin concentration was noted at 25 μg IP only.These changes may be related the secondary changes describedhistopathologically.

There were no other toxicologically significant changes.

8. Urinalysis

There were no apparent effects on urinalysis parameters.

9. Toxicokinetics

Plasma concentrations of MPP5 were generally similar between individualanimals of each group at each time point. Some variability in plasmaconcentration values was expected as dose levels were not normalizedaccording to body or prostate weight. MPP5 was not quantifiable insamples collected from control animals or from samples collected predose.

In test groups, plasma concentrations of MPP5 generally increased withincreasing dose levels of test article administered intraprostatically,with no detectable concentrations in any samples collected from animalsat 2 μg. MPP5 was detectable up to 24 hours in animals treated at 10 μg,8 hours in animals treated at 25 μg and 48 hours in Group 5 (25 μg,intravenous). In all, three composite profiles were obtained andconsidered for further evaluation.

A terminal phase could be estimated for the composite IV profile, wherethe terminal half-life was estimated as 12.8 hours. For the remainingprofiles, a terminal phase could not be estimated with confidence.Therefore, all parameters derived from k (t_(1/2), AUC_(0-inf) and %extrapolated) were not reported. For the IV profile, the percent ofAUC_(0-inf) extrapolated from AUC_(0-tlast) was less than 6%, indicatingthat this profile was well characterized from the experimental data.

Following intraprostatic dosing, observed t_(max) occurred at 4 hourspost dose for all cases, with peak levels of 2.95 and 3.51 ng/mL at 10and 25 μg, respectively. The observed AUC_(0-tlast) decreased at thehigher dose (48.5 vs 22.4 ng·h/mL). This result was biased, however, bythe shorter t_(last) observed at 25 μg IP (8 hours vs 24 hours at 10 μgIP). Following IV dosing the C_(max) could be back extrapolated to time0 hours with a value of 81.3 ng/mL. The observed peak value (at thefirst sampling time) was 74.3 ng/mL. The systemic clearance (CL) andvolume of distribution (V_(z)) was estimated as 46.3 mL/h and 841 mL,respectively.

Dose linearity following intraprostatic dosing was assessed at 10 to 25μg dose levels using dose normalized mean peak plasma concentration andarea under the curve exposure parameters (C_(max) and AUC_(0-tlast),respectively). Both dose normalized exposure parameters were decreasedfrom 10 μg to 25 μg. This could be indicative of limited absorption ofMPP5 into the systemic circulation from the prostate.

Based on the areas (AUC_(0-tlast)) observed in the mid to high dosesfollowing intraprostatic administration, the percent bioavailability wasdetermined by comparing these areas (normalized to dose level) to thedose normalized area obtained following intravenous administration. Theestimated bioavailabilities were 23.7 and 4.38%.

10. Organ Weights Intraprostatic Injection

Changes in absolute and relative organ weights (organ weight to bodyweight), occasionally attaining statistical significance were noted inthe prostate on all sacrifice days and in the spleen on sacrifice Day 2.Prostatic weights ranged from 26% to 45% higher in animals at 10 μg and25 μg on Day 2 compared to controls. Prostate weights were 16% to 24%lower compared to controls in treated animals on Day 15 and 19% to 21%lower at ≧10 μg on Day 29. These changes were consistent with theobserved microscopic findings. Spleen weights were approximately 36%lower at 25 μg on Day 2 compared to controls. This was considered asecondary change and recovered by Day 15.

Intravenous Injection

On Day 2, animals at 25 μg IV had higher liver and spleen weights ofapproximately 21% compared to controls without histological correlationand recovered by Day 15.

11. Gross Pathology MTD Study

Prostatic injection site changes were identified at all IP dose levels.Alterations included dark discoloration/area, mottling, andenlargement/swelling. Animals replaced on Day 1 (Nos. 6001, 8001) orfound dead (No. 8101) on Day 2 following IP injection had some of theprostatic injection site changes as described above. A specific cause ofdeath was not determined. Intravenous (IV) injection in the tail veinwas associated with scabs on the tail of Group 8 and 9 animals. Otherchanges were considered sporadic, procedure-related or agonal and nottreatment-related.

Main Study

Prostatic injection site changes were identified on all sacrifice days.Day 2 changes were characterized by dark discoloration/area/foci,adhesion, and enlargement/swelling at all dose levels. Day 15 and 29alterations were described as pale area, raised, firm, and/or small inanimals at ≧10 μg. Adhesions and pale areas were frequently noted on theliver and spleen on sacrifice Day 15 and 29 at ≧10 μg and were likelysecondary to the prostatic injection site alterations. Animal 4005 (25μg IP) found dead on Day 2 had some of the prostatic injection sitechanges as described above. A specific cause of death was notdetermined.

Intravenous injection in the tail vein was associated with scabs on thetail on all sacrifice days. Ulceration was present on Day 15 and 29 withor without loss of the tail tip. Liver enlargement was noted onsacrifice Day 2.

Other changes were considered sporadic, procedure-related or agonal andnot treatment-related.

12. Histopathology Intraprostatic Injection

Microscopic changes attributed to MPP5 were observed at the prostateinjection site at ≧2 μg on Day 2, 15 and 29. Minimal to severe acuteinflammation was observed at all doses on Day 2, generally, with adose-dependent increase in severity. The inflammation was characterizedby fibrin, mixed cell infiltrate and acinar necrosis. Edema andhemorrhage were observed in treated and control animals and therefore,considered partially procedure-related. Minimal to marked chronicinflammation and/or fibrosis were observed at all doses on Day 15 and29, generally, with a dose-dependent increase in the severity. Fibrosis,mononuclear infiltrate and acinar necrosis characterized theinflammation. These changes were frequently accompanied by concurrentacinar atrophy/dilatation. Minimal fibrosis and acinaratrophy/dilatation were infrequently observed in controls on Day 29 andtherefore, considered primarily treatment-related. Acute and chronicchanges were also observed in the adjacent prostatic lobe, generally,with a lower incidence and severity of change. Acute inflammation, edemaand hemorrhage correlated with higher prostate weights on Day 2 andchronic inflammation, fibrosis and acinar atrophy/dilatation with lowerprostate weights on Day 15 and 29 and generally correlated with themacroscopic findings. These alterations indicate an irritating effect ofMPP5 at the prostatic injection site.

Numerous microscopic observations in other tissues were consideredsecondary to the prostatic injection site inflammation either byexpansion into adjacent pelvic organs and throughout the abdominalcavity or systemic reactive and/or degenerative changes. Acute andchronic inflammation with or without hemorrhage and fibrosis of capsularor serosal surfaces, respectively, were observed in fat, liver, largeand small intestines, pancreas, spleen, stomach, seminal vesicles,epididymis, testis and urinary bladder. Reactive and/or degenerativechanges included: bone marrow myeloid hyperplasia; increasedextramedullary hematopoiesis in the spleen; lymphoid atrophy and/orhyperplasia in lymph nodes, spleen and thymus; hepatic mononuclearinfiltrate, single cell necrosis and increased mitotic figures, and;testicular atrophy (with concurrent epididymis oligo/aspermia). Spleniclymphoid atrophy would account for the low spleen weights noted in malesat 25 μg on Day 2 and therefore, considered secondary.

A specific cause of death was not identified for two males (25 μg) founddead on Day 2. However, it was assumed that the severity of theprostatic inflammation with its proximity to kidneys and concurrentsystemic degenerative alterations may have contributed to the death ofthese animals.

Other changes were sporadic, incidental, agonal or were expected in thisage and breed of rat and not directly treatment-related.

Intravenous Injection

Microscopic changes attributed to MPP5 were observed at the tail veininjection site on Days 2, 15 and 29. Minimal to marked acuteinflammation of the dermis and subcutis on Day 2 was characterized byfibrin, hemorrhage, necrosis and mixed cell infiltrate with or withoutepidermal ulceration and crust formation in the majority of animals. Oneanimal had moderate necrosis spreading to adjacent tissues and regionallymph nodes. A reduced incidence and severity of changes on Day 15 and29 suggested progressive recovery. Chronic inflammation characterized byfibrosis and mononuclear infiltrate was observed with or withoutepidermal ulceration and crust formation and infrequent necrosis andinflammation of the adjacent bone. Microscopic findings generallycorrelated with macroscopic alterations. These alterations indicate anirritating effect of MPP5, particularly in perivascular tissue at theinjection site.

A low incidence of changes in other tissues was considered secondary tothe injection site inflammatory changes observed on Day 2 and generally,recovered by Day 15 and 29. These included: necrosis and inflammation inthe spleen; perivascular neutrophil, mononuclear or mixed cellinfiltrate in the epididymis, seminal vesicles, testis and liver; bonemarrow myeloid hyperplasia; lymph node edema; lymphoid atrophy in lymphnodes, spleen and thymus, and; testicular atrophy observed on Day 29.Increased liver and spleen weights on Day 2 had no microscopiccorrelate.

5. CONCLUSION

In conclusion, the administration of MPP5 by single intraprostaticinjection at dose levels up to 40 μg or intravenous injection at doselevels up to 50 μg resulted in mortality at 25 μg and 40 μg IP with noclear cause of death, however the extent of the test article relatedprostatic inflammation and with proximity to the kidneys and concurrentsystemic degeneration were considered potential contributory factors tothe deaths. Mostly reversible changes were seen in clinical signs (≧2μg), hematology and clinical biochemistry parameters at ≧10 μg.Pathological changes persisted at all dose levels in a dose-relatedfashion but showed evidence of regression in animals treatedintravenously. Consequently, the no-observable-effect-level (NOEL) wasnot determined for either the intraprostatic or the intravenous route.

REFERENCES

-   Dunn, O. J. 1964, Multiple Comparisons using Rank Sums,    Technometrics, 6, 241-256.-   SAS Institute Inc., 1999.5AS/STAT® User's Guide, Version 8, Cary,    N.C.: SAS Institute Inc., 3884 pp.

Example 7 Acute Toxicity of MPP5 in Monkeys

This example shows preliminary results of a study indicating thatintraprostatic administration of a histidine-tagged MPP comprising a PSAcleavage site (MPP5) results in dose-dependent damage to the prostate.The objective of this study was to assess the potential localizedtoxicity of a single intraprostatic injection of MPP5 in sexually maturemale cynomolgus monkeys over a 2 week period.

Experimental Design Overview General Description:

A total of 16 male cynomolgus monkeys (Macaca fascicularis) wereassigned to treatment groups as shown in the Table 11 below. The animalswere approximately 3.6 to 11.7 years of age and weighed approximately2.8 to 7.9 kg. The animals were imported from China, Vietnam, Indonesiaand Mauritius.

TABLE 11 Group Number of Dose Level Number Sacrificed: No. Males (μg/gprostate¹) Day 3 Day 15 1 4 0 (control) 2 2 2 4 1 2 2 3 4 5 2 2 4 4 25 22 ¹Prostate weight will be estimated from a previously establishedrelationship between prostate weight and body weight. One-half of thedose will be injected into each lobe of the prostate.

All animals were dosed once under general anesthesia via perianalintraprostatic injection. The first day of dosing was designated Day 1.The animals were evaluated for changes in clinical signs (twice daily),food consumption (once daily), body weight (Days −1, 3, 8, and 15),electrocardiograms (prestudy and Days 2 and 14), and ophthalmiccondition (prestudy and Day 14). Clinical pathology indices (serumchemistry [including C-reactive protein], hematology and coagulation)were determined prestudy and on Days 3 and 14. Blood samples werecollected for toxicokinetic analysis, antibodies to the test article andprostate specific antigen (PSA) at various time points following doseadministration. Eight animals were euthanized on Days 3 and 15 asindicated in the Table 11. At termination, a full necropsy was conductedon all animals, and tissues were collected (including selectedperiprostatic tissues), preserved, processed and examinedmicroscopically. This study evaluated the acute localized toxicity ofMPP5.

The test article was MPP5, Lot No. PTIC-MF-PAL-DS-001 and the controlarticle was PBS-EDTA. A solution of the stock test article, in which theconcentration of the active ingredient was 3.2 mg/mL, was filteredthrough a suitable 0.22 micron PVDF filter prior to dose solutionpreparation on the day of preparation. Dilutions of the filtered stocktest article solution with the control vehicle were performed on the dayof dosing to yield a dosing solution at appropriate concentrations forachieving the intended doses.

Animals were housed as specified in the USDA Animal Welfare Act (9 CFR,Parts 1, 2 and 3) and as described in the Guide for the Care and Use ofLaboratory Animals (ILAR publication, 1996, National Academy Press).

Animals were initially assigned Provantis numbers that reflected theorigin of the monkey as shown in the table below. Animals assignedProvantis numbers of 6001-6008 were of Chinese origin. Animals assignedProvantis numbers of 7001-7004 were of Indonesian origin. Animalsassigned Provantis numbers of 8001-8003 were of Mauritius origin. Theanimal assigned Provantis number 9001 was of Vietnamese origin.

Due to the wide range of animal origins and bodyweights, the animalswere randomly assigned to treatment groups according to the table below.

Set A Set B Group Chinese/ Indonesian/ Chinese/ Indonesian/ No.Vietnamese Mauritius Vietnamese Mauritius 1 1 1 1 1 2 1 1 1 1 3 1 1 1 14 1 1 1 1

Test and control article administration, group assignments and doselevels:

Group Number of Dose Level Dose Volume Dose Solution No. Males (μg/gprostate¹) (μL/g prostate²) Conc. (μg/mL) 1 4 0 (control) 50 0 2 4 1 5020 3 4 5 50 100 4 4 25 50 500 ¹Prostate weight was estimated from apreviously established relationship between prostate weight and bodyweight. ²One-half of the dose was injected into each lobe of theprostate, that is, approximately 25 ul/g prostate per lobe.

Dosing was carried out as follows. The route of injection was Perianalintraprostatic bolus injection and the frequency was once. Monkeys wereinitially sedated with an intramuscular injection of ketamine and atemporary intravenous catheter was placed for administering sedativesand/or anesthetics during the surgical procedures. A small skin incisionwas made in the perianal region below the anus and muscle andsubcutaneous tissues were blunt dissected to allow visualization andidentification of the prostate gland. The test and control articles wereadministered on a prostate gland weight basis. Approximate weight of theprostate gland was estimated from the animal's body weight and apreviously established relationship between the body weight and prostategland weight (prostate weight (g)=0.07294+(−0.2309×kg)+(0.06296×kg²),where kg is body weight). The test and control articles wereadministered in approximately equal volumes to each of the left andright lobes of the prostate gland.

The perianal route was chosen because it is the most precise means ofadministering the test article directly to the prostate gland. The testarticle will also be administered locally to the prostate in humans.

Cage side observations: These were made twice daily (a.m. and p.m.),beginning at least 7 days prior to the day of dosing and continuingthrough the last day of sample collection. Each animal was observed forchanges in general appearance and behavior.

Food consumption was once daily, as part of the routine cage sideobservations, beginning at least 7 days prior to the day of dosing andcontinuing through the last day of sample collection (except as notedbelow). The number of biscuits remaining from the previous day's feedingwere observed. Exceptions to this procedure were for days of fasting forstudy procedures

Body weight measurements were taken prior to the first dose (Day −1),and on Days 3, 8 and 15 according to the following procedure. Food waswithheld before body weights were measured.

Electrocardiograms were recorded Prestudy, on Day 2 and 14 using Leads:I, II, III, aVR, aVL and aVF. Monkeys were temporarily restrained forthe procedure outside their cages in primate chairs, but were notsedated.

Ophthalmic Examinations were conducted by a veterinarian prestudy(within 3 weeks of Day 1) and on Day 14. Under light sedation withketamine, a direct opthalmoscope was used to examine the anterior andposterior chambers of the eye. A few drops of a mydriatic solution(typically 1% tropicamide) was instilled into each eye to facilitate theexamination.

Blood samples for evaluation of serum chemistry, hematology andcoagulation parameters were collected from all animals during Week −1and on Days 3 (prior to necropsy) and 14 (prior to ophthalmicexaminations). The animals were fasted for at least 8 hours (but notmore than 16 hours, without appropriate justification) prior to bloodcollections for serum chemistry.

Urine was collected for urinalysis by cage pan collection prestudy andon the morning following dosing (Day 2, approximately 24 hours afterdosing) and in terminated animals by cystocentesis at each necropsy(Days 3 and 15)

a) Serum Chemistry Collection Procedures

Method of Collection: Venipuncture—Any available vein, preferablyfemoral

TABLE 12 Serum Chemistry Parameters Sodium Calcium Potassium PhosohorusChloride Urea nitrogen (BUN) Carbon dioxide Creatinine Total bilirubin*Total protein Alkaline phosohatase (ALP) Albumin Lactate dehydrogenase(LDH) Globulin Aspartate aminotransferase (AST) Albumin/globulin ratioAlanine aminotransferase (ALT) Glucose Gamma-glutamyltransferase (GGT)Cholesterol C-Reactive Protein (CRP) Triglycerides *If suspected testarticle-related increases in total bilirubin occur, direct and indirectbilirubin concentrations will be determined.

b) Hematology

Blood samples were collected by venipuncture of any available vein,preferably femoral. The collection volume was 1 ml and the anticoagulantused was EDTA.

Parameters Analyzed:

TABLE 13 Hematology Parameters Red blood cell (RBC) count Meancorpuscular hemoglobin (MCH) White blood cell (WBC) count* Meancorpuscular volume (MCV) Hemoglobin concentration Mean corpuscularhemoglobin concentration (MCHC) Hematocrit Platelet counts Reticulocytecounts Blood cell morphology** *Includes total white blood cell,polysegmented neutrophil, band neutrophil, lymphocyte, monocyte,eosinophil, basophil, and other cell counts as appropriate. **The bloodsmear from all animals will be examined at each timepoint (includingprestudy).

c) Coagulation Parameters

Samples were collected by venipuncture of any available vein, preferablyfemoral. The collection volume was 1.8 mL and the anticoagulant wassodium citrate. The samples were processed to plasma and the followingparameters analyzed: Activated partial thromboplastin time (APTT),prothrombin time (PT), and fibrinogen.

d) Urinalysis

Samples were collected by the cagepan collection method (Prestudy and atapproximately 24 hours after dosing) and by cystocentesis; obtained atnecropsy. The collection volume was as available, up to 5 mL. Sampleswere processed according to standard procedures known in the art.

The following parameters were analyzed:

Urinalysis Parameters Color/Character Ketones pH Bilirubin Specificgravity Occult blood Protein Microscopics Glucose

F. Analysis Conducted of: 1. Toxicokinetic Samples

Samples were collected by venipuncture of any available vein, preferablyfemoral. Samples were taken prior to dosing and at 1, 2, 4, 8, 24 and 48hours postdose. The collection volume was 2 mL and no anticoagulant wasused. Samples were processed to serum.

Sera were divided into two aliquots of approximately equal volume. Eachsample was labeled with the animal number, dose group, day ofcollection, date, nominal collection time, study number and aliquotnumber. Samples were stored at approximately −70° C., and were analyzedfor MPP5 concentration by ELISA.

2. Antibody Samples

Samples from all available groups/animals were tested. Samples werecollected by venipuncture of any available vein, preferably femoral,prior to dosing and on Day 14. The collection volume was 2 mL. Noanticoagulant was used. Samples were processed to serum. Samples werestored at approximately −70° C.

Prostate Specific Antigen Analysis

Samples from all available groups/animals were tested. Samples werecollected by venipuncture of any available vein, preferably femoral,pior to dosing and on Days 2, 3, and 10. The collection volume was 2 mL.No anticoagulant was used. Samples were processed to serum and stored atapproximately −70° C.

Terminal Procedures and Anatomic Pathology

Termination: The animals were terminated by exsanguination while underdeep anesthesia induced with ketamine and Beuthanasia®-D or equivalent.Food rations were withheld overnight prior to the day of sacrifice. Theanimals were sacrificed according to the following schedule:

Group Day 3, Set B, Day 15, Set A, No. No. of Males No. of Males 1 2 2 22 2 3 2 2 4 2 2

Final Body Weight: A terminal body weight was obtained at necropsy forall scheduled and unscheduled sacrifices. This body weight was used tocalculate organ/body and organ/brain weight ratios.

Gross Necroscopy: A complete gross necropsy was conducted on all animalsfound dead or sacrificed during the study (both scheduled andunscheduled sacrifices). The necropsy included examination of: Carcassand musculoskeletal system, all external surfaces and orifices, cranialcavity and external surface of the brain, neck with associated organsand tissues, thoracic, abdominal and pelvic cavities with theirassociated organs and tissues.

Urine samples: Urine (as available to a maximum of 5 mL) was collectedfrom the bladder at necropsy and analyzed as described in the ClinicalPathology section of this protocol.

Organ weights: The following organs (when present) were weighed beforefixation.

Paired organs will be weighed together unless gross abnormalities arepresent, in which case they will be weighed separately. The pituitarywas weighed post fixation.

Organs Weighed Adrenals Brain Epididymides Heart Kidneys Liver LungsPituitary (post fixation) Prostate (without seminal vesicles) SpleenTestes Thymus Thyroid with parathyroids

Organ/body weight ratios were calculated (using the final body weightobtained prior to necropsy), as well as organ/brain weight ratios.

Tissue collection and preservation: The following tissues and organs (orportions of), were collected and preserved in neutral-buffered 10%formalin (except for the eyes, which were preserved in Davidson'sfixative for optimum fixation).

Tissues Collected Cardiovascular Urogenital Aorta Kidneys Heart UrinaryBladder Digestive Testes Salivary Gland (mandibular) Epididymides TongueProstate Esophagus Periprostate Tissues Stomach Anal Sphincter MuscleSmall Intestine Bladder adjacent to Prostate Duodenum Prostatic UrethraJejunum Seminal Vesicles Ileum Ureters Large Intestine Vas DeferensCecum Endocrine Colon Adrenals Rectum Pituitary PancreasThyroid/Parathyroids^(a) Liver Skin/Musculoskeletal Gall bladder SkinRespiratory Bone (femoral head) Trachea Bone (7th rib) Lung SkeletalMuscle (psoas and diaphragm) Lymphoid/Hematopoietic Nervous/SpecialSense Bone Marrow (sternum) Eyes with Optic Nerve Thymus Sciatic NerveSpleen Brain Lymph Nodes Spinal Cord (thoracic) Inguinal OtherMesenteric Animal Number Tattoo Gross Lesions ^(a)The occasional absenceof the parathyroid gland from the routine tissue section will notrequire a recut of the section.

Histopathology: For all animals necropsied, the tissues listed in thetable above (except tattoos) were embedded in paraffin, sectioned,stained with hematoxylin and eosin, and examined by a VeterinaryPathologist certified by the ACVP.

Statistical Analyses

Group means and standard deviation values were calculated for allnumerical data obtained by Sierra, including body weights, clinicalpathology parameters (excluding non-numerical data), and organ weightdata.

Further statistical analyses were performed with the SAS® System,Version 8.1. Significant intergroup differences will be evaluated by useof an analysis of variance (ANOVA), followed by a multiple comparisonstest. The assumptions that permit use of a parametric ANOVA will beverified using the Shapiro-Wilkes test for normality of the data andLevene's test for homogeneity of variance, with p≦0.001 level ofsignificance required for either test to reject the assumptions. If bothassumptions are fulfilled, a single-factor ANOVA will be applied, withanimal grouping as the factor, utilizing a p≦0.05 level of significance.If the parametric ANOVA is significant at p≦0.05, Dunnett's test will beused to identify statistically significant differences between thecontrol group and each test article-treated group at the 0.05 level ofsignificance. If either of the parametric assumptions is not satisfied,then the Kruskal-Wallis non-parametric ANOVA procedure will be used toevaluate intergroup differences (p≦0.05). The Dunn's multiple comparisontest will be applied if this ANOVA is significant, again utilizing asignificance level of p≦0.05.

Preliminary Results

Prostate: All treated animals had lesions in their prostates (See FIGS.32C-H and 33C-H). Group 1 (Control) animals had minimal inflammation,hemorrhage, and fibrosis on Day 3 and fibrosis on Day 15 consistent withreaction to an injection, and healing. There were significant severelesions in Groups 3 and 4 (FIGS. 32E-32H, FIGS. 33 E-H) and 33F). Therewas no difference in extent or severity of lesions between Groups 3 (5μg/g prostate) and 4 (25 μg/g prostate). There were significant, lesssevere lesions in Group 2 (1 μg/g prostate). (FIGS. 32C, 32D, 33C, 33D).

On Day 3 the primary reaction to the test article was coagulativenecrosis of large parts of the prostate with extensive hemorrhage, andmixed cell inflammation. Inflammation was primarily neutrophilic withlesser numbers of eosinophils, lymphocytes and macrophages. In someareas there was liquefactive necrosis. Coagulative necrosis was mild inGroup 2 and moderate to marked in Groups 3 and 4.

On Day 3 in Group 2, extensive repair was underway with markedregenerative hyperplasia of gland epithelium that progressed to squamousmetaplasia, at the margins of areas of coagulative necrosis, and mild tomarked activation and proliferation of interstitial cells. On Day 3 inGroups 3 and 4 repair was just beginning evidenced by mild activationand proliferation of interstitial cells.

On Day 15 in Group 2 necrosis and hemorrhage had resolved. Cavitation ofthe prostate was not noted. The lesions had fibrosed in with minimal tomild ongoing regenerative hyperplasia and squamous metaplasia of glands.Inflammation was primarily macrophages and lymphocytes with lessernumbers of polymorphonuclear leukocytes. In one animal, these wereprimarily eosinophils.

On Day 15 in Groups 3 and 4, there was ongoing coagulative necrosis andhemorrhage with more severe liquefactive necrosis and cavitation of theprostate, and ongoing mixed cell inflammation. Repair in these groupsconsisted of moderate to marked fibrosis and fibroplasia of theinterstitium at the margins of necrotic lesions, with regenerativehyperplasia progressing to squamous metaplasia of glands. Inflammationwas primarily neutrophilic in areas with ongoing necrosis but had arelatively greater percentage of lymphocytes and macrophages at themargins of lesions in areas of fibrosis.

Periprostatic Tissues:

Seminal vesicles in ⅚ treated animals on Day 3 had minimal to moderatemineralization of the secretion in the gland lumen. This is seenoccasionally as a background finding, but not to this extent or asfrequently as seen here. Therefore this was likely secondary to changesin the prostates. On Day 15 the only affected animal was a control.

Prostatic urethras had some inflammatory cell infiltrates, likelysecondary to changes in the prostates.

Example 8 Activation of MPP5 by Prostate Tissue

The ability of extracts from prostates of various animals to activate anMPP according to the present invention was examined. An in vitro studywas performed in which extracts of rat, dog, monkey, and human prostatetissues were incubated with MPP5 to determine percent activation of theMPP.

The experimental protocol was as follows. Fresh prostate tissue wasobtained from a Sprague Dawley rat and a single beagle dog. Frozenprostate tissues were obtained from a single Cynomolgus monkey and froma single human. Human prostate tissue was obtained as archived researchmaterial from Johns Hopkins University IRB approved clinical study. Forthis analysis, prostates were sectioned (˜100-500 mg pieces) andsuspended in serum-free RPMI 1640 cell culture media at a concentrationof equal volume of media per volume of tissue. Tissue samples wereincubated in this media at 37° C. for 2 hours. After centrifugation,supernatant was frozen at −80° C.

Hemolysis assay: Samples were thawed at 37° C., centrifuged, and thesupernatants collected. Protein concentration of each supernatant wasdetermined using the Bradford Assay. Samples were diluted to the samestarting protein concentration. Aliquots of supernatant from monkey andhuman samples were obtained for PSA determination using a standard ELISA(Hybritech®, Beckman Coulter) methodology. A solution of 2% fresh humanred blood cells (RBCs) suspended in phenol red-free Hanks Buffered SaltSolution (HBSS) was prepared each day. Red blood cells were pelleted,resuspended in three volumes of HBSS to remove excess serum andresuspended to produce a 50% solution in phenol red-free HBSS. Toprepare test samples, a 50% RBC sample was gently vortexed to suspendRBCs. Aliquots of RBCs were added to 230 μL phenol red-free HBSS toproduce a 4% (v/v) solution. To this suspension an aliquot of 240 μL ofprostate section conditioned RPMI media was added, and subsequently, a10 μl, aliquot of MPP5 from stock of 100 μg/mL was added so that totalMPP5 added per assay was 1 μg and final volume of assay was 500 μL. ThisRBC/MPP5/Tissue solution was incubated for 1 hr at room temperature.Samples were then centrifuged to pellet non-lysed RBCs and 100 μLaliquot of supernatant from each sample was obtained and immediatelymeasured spectrophotometrically at 540 nm for hemoglobin release due toRBC lysis. Controls included sham-treated RBCs (negative control) andRBCs lysed with 1% Triton-X100 (positive control). In order to comparethe extent of hydrolysis, serial dilutions of each sample of prostatetissue-conditioned media were assayed. Dilutions of extract of 1:1, 1:2,1:4, 1:8, 1:16, and 1:32 were used in this study. All samples wereassayed for hemolysis in triplicate.

The results indicated that the human prostate tissue was most active incleaving MPP5, while rat and monkey prostate tissues produced a lowerresponse, whereas dog prostate tissue did not show any activity towardsMPP5 (FIG. 36). Although the rat lacks the PSA gene, it has been shownto possess an S3 kallikrein homolog to human PSA (Onozawa et al., 2001).This PSA-like protein, identified in the rat ventral prostate, showsnucleotide and amino acid sequence homology of 64% and 49%,respectively, with human PSA. Furthermore, the rat S3 kallikrein andhuman PSA have similar isoelectric points and molecular weights. Thus,it is likely that MPP5 is activated in rat prostate tissue by thisPSA-like S3 kallikrein.

Lack of activation by the dog prostate is consistent with theobservation that the dog does not possess the PSA gene.

Example 9 Activation of MPP5 by Plasma/Serum

The ability of serum from human, monkey, dog, rat or mouse to cleaveMPP5 was determined as follows.

MPP5 (1.073 mg/mL) was thawed on ice at 4° C., aliquoted, and refrozenat −80° C. Two assays were performed for each condition. 5 μg MPP5 wasincubated in 20 mM HEPES buffer (pH 7.4) containing 150 mM NaCl and 25μL human, monkey, dog, rat or mouse serum at 37° C. for 10 minutes in atotal volume of 250 μL. In control experiments, 25 μg chymotrypsin wasadded to the reaction mixture before addition of serum (positivecontrol).

In other control experiments serum was replaced with an equal volume ofbuffer (negative control). The reaction was stopped by addition of 5 μLof 100 mM PMSF in isopropyl alcohol followed by cooling on ice. A 15 μLaliquot of stopped reaction mixture was added to an equal amount of 2×BioRad sample loading buffer containing 0.5% β-mercaptoethanol andheated at 95° C. for 5 min to denature all proteins and blockprotein-protein interactions. A 5 sample was electrophoresed on pre-cast4-12% Bis-Tris gels (Invitrogen) using XT MOPS running buffer (BioRadLaboratories) at 100 V for 60 minutes. Proteins in the gels weretransblotted onto nitrocellulose (BioRad Laboratories) and the membraneblocked with non-fat milk (5% in tris-buffered saline with 0.1% Tween 20(TBST) for 1 hour at room temperature. MPP5 was detected by incubatingthe membranes in purified polyclonal rat anti-MPP5 at a dilution of1:250,000 in TBST for 1 hour at room temperature. After washing threetimes with TBST, the blot was incubated in HRP-linked goat anti-ratantibody (Jackson ImmunoResearch) at a dilution of 1:20,000 in TBST.Antibody binding to the membrane was detected using chemiluminescenceaccording to the kit manufacturer (Cell Signaling) and recordedreal-time using an Alpha Innotech Fluor Chem SP with chemiluminescenceautoexposure settings to avoid saturation using a 4 megapixel CCDcamera. Blots were quantified densitometrically using a voxel basedprogram (ImageQuant software; Alpha Innotech, San Leandro, Calif.).Percent of cleaved protein remaining was determined for each lane bydividing density of the cleaved band by the sum of the intact andcleaved bands, after correction for background. Percent cleaved was thencompared to no-serum and serum plus chymotrypsin controls.

Estimation of percent cleavage of MPP5 was accomplished byelectrophoresis and densitometric quantification of western blots. Thepercent cleaved was then compared to MPP5 only (negative) and MPP5+chymotrypsin (positive) controls to determine whether MPP5 is cleaved byserum enzymes. Under these experimental conditions, cleavage of MPP5 byhuman, monkey, dog, rat or mouse serum was not detectable. Table 14shows the percentage of MPP5 that is cleaved after incubation withvarious sera. FIG. 37 shows western blots of MPP5 after incubation for10 minutes in the absence or presence of various sera. Panel A showsMPP5 incubated with either 25 μL serum from human males (H) (lanes 2-5)or Cynomolgous monkey (Mk) (lanes 6-7) in 250 μL assay volume. Lane 3contains MPP5, human serum and chymotrypsin, but was not incubated. Lane8 is molecular weight marker. (B) MPP5 incubated with either 25 μL serumfrom Mouse (Mu) (lanes 2-5), Dog (D) (lanes 6-7) or Rat (R) (lanes 8-9)in 250 μL assay volume. Lane 3 contains MPP5, human serum andchymotrypsin, but was not incubated. Lane 10 is molecular weight marker.

TABLE 14 Species Comparison of Percent Cleavage of MPP5 in Serum BufferHuman Monkey Dog Rat Mouse Percent 1.45 ± 1.08 0.90 ± 0.31 1.78 ± 1.511.20 ± 1.0 1.87 ± 0.32 1.33 ± 0.10 Cleaved

These results suggest that MPP5 is not activated in normal human serumand also suggest that MPP5 would not become activated in the event ofleakage into the blood following intraprostatic injection even in menwith extraordinarily high levels of serum PSA. These results areconsistent with published data demonstrating that PSA is enzymaticallyinactivated in the blood by serum protease inhibitors, primarilyα1-antichymotrypsin and α2-macroglobulin (Lilja et al., 1991; Otto etal., 1998).

Example 10 Activation Of MPP5 By Non-PSA Proteases

An in vitro study was performed to determine the sensitivity of MPP5 tonon-PSA proteases that the prodrug could potentially encounter if it wasinadvertently exposed to tissues outside of the prostate. Specifically,several common proteases including PSA, furin, trypsin, chymotrypsin,thrombin, MMP-7, cysteine protease cathepsin B, and the serine proteaseshK1, hK2, and uPA were evaluated for their potential to cleave MPP5.

The assays were carried out as follows. Native proaerolysin (wt PA; 0.84mg/mL) and MPP5 at 1.073 mg/mL were used for assays testing allproteases except for assay #2 with furin, in which MPP5 of Lot #N-PTIC-MF-PAL-BX; at 1 mg/ml was used.

To measure activation by PSA cleavage, 5 μg of native proaerolysin orMPP5 were incubated in 20 mM HEPES buffer (pH 7.4), containing 150 mMNaCl. Various amounts of PSA were added (0-10 μg PSA according to alogarithmic scale) and incubated at 37° C. for 60 minutes in a totalvolume of 250 μL. The reaction was stopped by addition of 5 μL of 100 mMPMSF in isopropyl alcohol followed by cooling on ice. A 15 μL aliquot ofstopped reaction mixture was added to an equal amount of 2× BioRadsample loading buffer containing 0.5% (3-mercaptoethanol and heated at95° C. for 5 minutes. The sample was electrophoresed on pre-cast 10%Tris-HCl gels using XT MOPS running buffer (BioRad Laboratories) at 200V for 30 minutes. The proteins were detected by silver staining.

To measure activation by furin cleavage in study #1, five μg of NativePA or MPP5 were incubated in 20 mM HEPES buffer (pH 7.4) containing 150mM NaCl and various amounts of furin (0-3.2 ng of furin according to alogarithmic scale) at 37° C. for 10 minutes in a total volume of 250 μL.The reaction was stopped by addition of 5 μL of 100 mM PMSF in isopropylalcohol followed by cooling on ice. A 15 μL aliquot of stopped reactionmixture was added to an equal amount of 2× BioRad sample loading buffercontaining 0.5% β-mercaptoethanol and heated at 95° C. for 5 minutes.The sample was electrophoresed on pre-cast 10% Tris-HCl gels using XTMOPS running buffer (BioRad Laboratories) at 200 V for 30 minutes. Theproteins were detected by silver staining.

To measure activation by furin cleavage in study #2, five μg of nativePA or MPP5 were incubated in 20 mM HEPES buffer (pH 7.4) containing 150mM NaCl, 1 mM CaCl₂ and 0 to 3 units of furin at 37° C. for 60 minutesin a total volume of 250 μL. Note that in earlier experiment (Furin,Study # 1), the incubation time was 10 minutes at the same enzymeconcentration. The reaction was stopped by addition of 2.5 μL of 100 mMPMSF in ethanol followed by cooling on ice. A 15 μL aliquot of stoppedreaction mixture was added to an equal amount of 2× BioRad sampleloading buffer containing 0.5% β-mercaptoethanol and heated at 95° C.for 5 minutes. The sample was electrophoresed on pre-cast 10% NovexBis-Tris Nupage gels (Invitrogen) using 1×MOPS-SDS running buffer at200V for 50 minutes. The proteins were detected by silver staining.

To measure activation by chymotrypsin, 5 μg of native PA or MPP5 wereincubated in 20 mM HEPES buffer (pH 7.4) containing 150 mM NaCl andvarious amounts of chymotrypsin (0-500 ng chymotrypsin according to alogarithmic scale) at 37° C. for 10 minutes in a total volume of 250 μL.The reaction was stopped by addition of 5 μL of 100 mM PMSF in isopropylalcohol followed by cooling on ice. A 15 μL aliquot of stopped reactionmixture was added to an equal amount of 2× BioRad sample loading buffercontaining 0.5% β-mercaptoethanol and heated at 95° C. for 5 minutes.The sample was electrophoresed on pre-cast 10% Tris-HCl gels using XTMOPS running buffer (BioRad Laboratories) at 200 V for 30 minutes. Theproteins were detected by silver staining.

To measure activation of MPP5 by thrombin in Study #1, 5 μgaerolysin-related protein (native PA or MPP5) was incubated in 20 mMHEPES buffer (pH 7.4) containing 150 mM NaCl and various amounts ofthrombin (0-12 μg of 0.23 Unit/μg thrombin according to a logarithmicscale) at 37° C. for 10 minutes in a total volume of 250 μL. Thereaction was stopped by addition of 5 μL of 100 mM PMSF in isopropylalcohol followed by cooling on ice. A 15 μL aliquot of stopped reactionmixture was added to an equal amount of 2× BioRad sample loading buffercontaining 0.5% (3-mercaptoethanol and heated at 95° C. for 5 minutes.The sample was electrophoresed on pre-cast 10% Tris-HCl gels using XTMOPS running buffer (BioRad Laboratories) at 200 V for 30 minutes. Theproteins were detected by silver staining.

To measure activation of MPP5 by thrombin in Study #2, two thrombindilutions, 1/66 and 1/25, were made in the thrombin dilution bufferprovided with the thrombin kit (Novagen) used in these experiments. Tworeaction mixtures were prepared containing 10 μg of MPP5(N-PTIC-MF-PAL-BX) in 1× cleavage buffer as provided with the thrombinkit. Two reaction mixtures containing native proaerolysin with a His tag(PA-EndHis) were prepared the same way. Thrombin was added to one of thePA-EndHis mixtures at 0.15 units and to one of the MPP5 mixtures at 0.4units. Total incubation volume was 50 μl in each case. The reactionmixtures were incubated at room temperature for 6.5 hours, and this wasfollowed by inhibition of proteolysis by the addition of phenylmethylsulfonyl fluoride (Sigma) to a final concentration of 1 mM. The sampleswere stored overnight on ice at 4° C. They were then prepared in 1×LDSsample buffer (Invitrogen) and heated at 70° C. for 10 minutes beforebeing loaded and run on a 10% Bis-Tris NuPAGE gel (Invitrogen) undernon-reducing conditions at 200 V constant voltage for 50 minutes in1×MOPS-SDS running buffer. The proteins were detected by silverstaining.

To measure activation by trypsin, 5 μg aerolysin-related protein (wt PAor MPP5) was incubated in 20 mM HEPES buffer (pH 7.4) containing 150 mMNaCl and various amounts of Type I trypsin (0-500 ng Trypsin accordingto a logarithmic scale) at 37° C. for 10 minutes in a total volume ofμL. The reaction was stopped by addition of 5 μL of 100 mM PMSF inisopropyl alcohol followed by cooling on ice. A 15 μL aliquot of stoppedreaction mixture was added to an equal amount of 2× BioRad sampleloading buffer containing 0.5% (β-mercaptoethanol and heated at 95° C.for 5 minutes. The sample was electrophoresed on pre-cast 10% Tris-HClgels using XT MOPS running buffer (BioRad Laboratories) at 200 V for 30minutes. The proteins were detected by silver staining.

To measure activation by uPA, 5 μg of PA and MPP5 were incubatedseparately in 20 mM HEPES buffer (pH 7.4) containing 150 mM NaCl and10-0.16 μg of uPA at 37° C. for 4 hours in a total volume of 250 μL. Thereaction was stopped by addition of 5 μL of 100 mM PMSF in isopropylalcohol followed by cooling on ice. A 15 μL aliquot of stopped reactionmixture was prepared in 1× sample buffer (Invitrogen) containing 0.5%2-mercaptoethanol and heated at 95° C. for 5 min. The sample (100 ng)was electrophoresed on pre-cast 10% Novex Bis-Tris NuPAGE gels(Invitrogen) using 1×MOPS-SDS running buffer under reducing conditionsat 200V for 50 minutes. The proteins were detected by silver staining.

To measure activation by cathepsin B, 5 μg of PA and MPP5 were incubatedseparately in 20 mM HEPES buffer (pH 7.4) containing 150 mM NaCl, 1 mMEDTA, 5 mM L-cysteine and 24-0.375 units of cathepsin B at 37° C. for 4hours in a total volume of 250 μL. The reaction was stopped by additionof leupeptin to a final concentration of 2 μM (Sigma method), followedby cooling on ice. A 15 μL aliquot of stopped reaction mixture wasprepared in 1× sample buffer (Invitrogen) containing 0.5%2-mercaptoethanol and heated at 95° C. for 5 min. The sample (100 ng)was electrophoresed on pre-cast 10% Novex Bis-Tris NuPAGE gels

(Invitrogen) using 1×MOPS-SDS running buffer under reducing conditionsat 200V for 50 minutes. The proteins were detected by silver staining.

To measure activation by MMP-7, 5 μg of PA and MPP5 were incubatedseparately in 20 mM HEPES buffer (pH 7.4) containing 150 mM NaCl, 10 mMCaCl₂ and 1.5-0.0234 μg of MMP-7 at 37° C. for 3 hours in a total volumeof 250 μL. The reaction was stopped by addition of 8.4 μL of 31 mM1,10-phenanthroline monohydrate (1 μM final) in ethanol followed bycooling on ice. A 15 μL aliquot of stopped reaction mixture was preparedin 1× sample buffer (Invitrogen) containing 0.5% 2-mercaptoethanol andheated at 95° C. for 5 min. The sample (100 ng) was electrophoresed onpre-cast 10% Novex Bis-Tris NuPAGE gels (Invitrogen) using 1×MOPS-SDSrunning buffer under reducing conditions at 200V for 50 minutes Theproteins were detected by silver staining.

To measure activation by hK1, 5 μg of hK1 was activated by 0.05 μgthermolysin in a final volume of 50 μl of TCN buffer (50 mM Tris, 10 mMCaCl₂, 0.15 M NaCl, pH 7.5), incubated at 37° C. for 1 hour andinhibited with 2.5 μl 200 mM 1,10-phenanthroline monohydrate in 95%ethanol (R & D Systems method). One μg of PA and PSA-PAH1 were incubatedseparately in 20 mM HEPES buffer (pH 7.4) containing 150 mM NaCl, 1 mMEDTA, and 1-0.015625 μg of activated hK1 at 37° C. for 4 hours in atotal volume of 50 μL. The reaction was stopped by addition of PMSF inisopropyl alcohol to a final concentration of 2 mM, followed by coolingon ice. A 5 μL aliquot of stopped reaction mixture was prepared in 1×sample buffer (Invitrogen) containing 0.5% 2-mercaptoethanol and heatedat 95° C. for 5 min. The sample (100 ng) was electrophoresed on pre-cast10% Novex Bis-Tris NuPAGE gels (Invitrogen) using 1×MOPS-SDS runningbuffer under reducing conditions at 200V for 50 minutes. The proteinswere detected by silver staining.

To measure activation by hK2, 1 μg of PA and MPP5 were incubatedseparately in 20 mM HEPES buffer (pH 7.4) containing 150 mM NaCl and0.25-0.0039 μg of hK2 at 37° C. for 1 hour in a total volume of 50 μL.The reaction was stopped by addition of PMSF in isopropyl alcohol to afinal concentration of 2 mM, followed by cooling on ice. A 10 μL aliquotof stopped reaction mixture was prepared in 1× sample buffer(Invitrogen) containing 0.5% 2-mercaptoethanol and heated at 95° C. for5 min. The sample (100 ng) was electrophoresed on pre-cast 10% NovexBis-Tris NuPAGE gels (Invitrogen) using 1×MOPS-SDS running buffer underreducing conditions at 200 V for 50 minutes. The proteins were detectedby silver staining.

Results of this study indicated that the sensitivity profiles betweenMPP5 and proaerolysin (PA) are very different, with native proaerolysin(PA) being more sensitive to the range of proteases (except PSA) thanMPP5 (Table 15).

TABLE 15 In Vitro Protease Sensitivity Study Results Amount RequiredAmount Required for 50% Cleavage for 50% Cleavage of Specific ActivitySpecific Activity Protease Name of MPP5 Native Proaerolysin Towards MPP5Towards Proaerolysin Furin >3.2 ng 1.2 ng 0.24 nM/μg/min 9.69 nM/μg/minTrypsin 813 ng 9.5 ng 5,700 nM/μg/min 489,000 nM/μg/min Chymotrypsin 31μg 3.0 μg 150 nM/μg/min 1550 nM/μg/min Thrombin >12 μg >12 μg<0.00000078 nM/μg/min <0.00000078 nM/μg/min MMP-7 Inactive Inactive 0 0Cathepsin B 170 units 51.6 units 1.2 fmol/μg/min 3.9 fmol/μg/min hK1Inactive 2.63 μg 0 15.3 fmol/μg/min hK2 Inactive 0.1 μg 0 1.61pmol/μg/min Prostate-specific 12.2 μg 49.7 μg 0.0635 nM/μg/min 0.0156nM/μg/min antigen (PSA), also referred as hK3 uPA 173 μg 7.79 μg 1.1fmol/μg/min 25.8 fmol/μg/min Note: Units reported reflect those asrecorded in the raw data.

Example 11 Biodistribution of MPP5 in RATS

In order to establish the biodistribution of MPP5 in the prostate andpotential distribution to surrounding tissues following single-doseintraprostatic administration, a radiolabeled quantitative whole bodyautoradiography study using ¹²⁵I-MPP5 was performed in maleSprague-Dawley rats. The radioactivity concentration (±S.D.) in the doseformulation was 1.6×10⁹±44.02×106 dpm/g (730.94 μC/g). Based on thestandard deviation and the coefficient of variation around the meanconcentration value, the dose formulation was considered homogeneous.The mean dose of formulated ¹²⁵I-MPP5 administered by injection into theprostate gland was 8.73 μg/animal (5.86 μCi/animal in a volume of 10 μL.

Duplicate aliquots of blood (2×50 μL) were sampled for radioactivityanalysis. Blood was centrifuged at 3500 rpm and 4° C. for approximately10 minutes (within 60 minutes of collection) and duplicate aliquots ofplasma (2×50 μL) were sampled for radioactivity analysis. Duplicateweighed aliquots of whole blood were solubilized (Soluene-350) anddecolorized with hydrogen peroxide (30% w/v) prior to mixing with liquidscintillation fluid for radioactivity measurement. Duplicate aliquots ofplasma were mixed directly with liquid scintillation fluid forradioactivity measurement.

For quantitative whole body autoradioluminography, animals were deepfrozen in a mixture of hexane and dry ice for 20 minutes. Animals werethen embedded lying on their right side in a 2% CMC medium using afreezing frame according to Standard Operating Procedures in order tocollect sagittal whole-body sections. Twelve holes were made in eachfrozen CMC block in order to incorporate ten ¹²⁵I standard solutions andthe two quality control solutions. Blood spiked with ¹⁴C or ¹²⁵I wereinserted in four drilled holes of each CMC block, which were used, ifrequired, as reference dots for identification of structures presentinga low radioactivity level or low contrast. Each animal specimen blockwas sectioned using the Leica CM 3600 cryomicrotome. 30 μm sections werecollected and identified the animal no., time point, section no.,section date and knife position.

The results indicated that following a single-dose intraprostaticadministration, the concentration of radioactivity in the blood andplasma were low, suggesting little apparent absorption followingintraprostatic administration. The highest concentration ofradioactivity (9.445 μg Eq/g) was obtained at the first sampling timepoint (3 h) from the right ventral prostate injection site. High levelsof radioactivity were also observed in other lobes of the prostate (leftventral, and right and left dorsal lobes) but decreased over time to thefinal sampling point of 96 h. At this final time point, theconcentration of radioactivity in the prostate was low for all areas ofthe prostate except the right ventral prostate injection site (0.268 μgEq/g). Other than the prostate, only the bladder and the thyroidexhibited radioactivity concentrations higher than either blood orplasma. Thyroid levels of radioactivity (¹²⁵I) increased over time from12 to 48-h post dose and remained elevated until the final time point at96-h post treatment. Sequestration of ¹²⁵I in the thyroid may beindicative of free ¹²⁵I distributing to the thyroid. Low concentrationsof radioactivity were observed in the adrenal gland (≦0.034 μg Eq/g),kidney (≦0.032 μg Eq/g), liver (≦0.045 μg Eq/g), lung (≦0.041 μg Eq/g)and pancreas (≦0.022 μg Eq/g). The brain exhibited the lowestconcentration of radioactivity (≦0.003 μg Eq/g). Extremely low levels ofradioactivity were noted in all other major organs at all times,suggesting that significant systemic distribution did not occur. Tissueto plasma levels increased over time, suggesting that the MPP5 wascleared faster from plasma than from tissues. Therefore, thisbiodistribution study suggests that MPP5 remains largely at the localsite of administration, with only limited peripheral distribution andtoxicity to surrounding cells.

Example 12 Toxicokinetics of MPP5 in Monkeys

The toxicokinetics of MPP5 were also established followingintraprostatic administration in sexually mature male Cynomolgus monkeysas described in Example 7. Four monkeys per group were dosedintraprostatically with control saline or 0.35, 4.14, or 25.79 μg MPP5/gprostate tissue using 2×25 μL injections (25 μL/lobe). Blood sampleswere obtained from all monkeys (16) prior to dose and at 1, 2, 4, 8, 24,and 48-h post-dose. Preliminary results of this study are also describedin Example 7. The following represents the finalized toxicokineticresults of this study.

Study samples were analyzed for MPP5 using a validated ELISA method. Thelower limit of quantitation (LLOQ) of the ELISA method was 5 ng/mL using50 μL serum in duplicate analysis. No appreciable systemic levels ofMPP5 were detected following intraprostatic administration. One animalin Group 2 presented concentrations over the LLOQ (5 ng/mL) for all timepoints. This was considered unusual compared to animals from the samedose group, and all samples from this animal were repeated in asubsequent assay for confirmation. All original results were confirmedin the repeat analysis. As the pre-dose sample was also over the LLOQ,it was determined that the observed concentrations in this animal werelikely due to matrix interference and were not treatment related.

Example 13 Evaluation of Prostate Morphology of MPP5 Treated Monkeys

Further analyses of sections of monkey prostate collected in the studydescribed in Examples 7 and 12 were conducted. Hematoxylin & Eosin (H&E)staining was performed to examine morphology of treated prostate,immunohistochemical staining for PSA was performed to examinedistribution of PSA, and MPP5 staining was performed in order to examinedistribution of MPP5. The protocols used are described following.

Materials and Reagents: 96 slides (6 per monkey) of sections fromcontrol and MPP5 treated monkey prostate from were stored in a sealedcontainer at room temperature. Sections of prostate tissue from monkeysdosed intraprostatically with vehicle or 0.35, 4.1 or 25 μg MPP5/gram ofprostate were prepared and stained with H&E. Sections were alsoimmunohistochemically stained for PSA and MPP5 according to methodsknown in the art.

Image analysis: Histological sections of monkey prostate were evaluatedusing 1.25× objective. Metamorph™ software package (Molecular Devices,Sunnyvale, Calif.) was used to outline the total area of the prostategland and the total area of MPP5 induced injury. This software providestotal area as number of pixels. A 1×1 cm square was placed on each slideas a standard to determine number of pixels/cm². The area of damage fromeach dose of MPP5 was then determined and converted into cm² of damage.Percent area of damage was determined by ratio of injured area/totalprostate area multiplied by 100.

Analysis of control (normal) monkey prostate demonstrated that theCynomolgus monkey prostate is similar to the human prostate in terms ofglandular morphology, and distribution of PSA is restricted to columnarepithelial cells lining the ducts. The monkey prostate gland, like thehuman, surrounds the urethra. Therefore, the monkey prostate representsthe best available animal model for studying activation and toxicity ofthe PSA-activated protein toxin, MPP5, when injected intraprostatically.

Morphological characterization of the prostate tissue and distributionof PSA and MPP5 in the prostates from this study showed a dose-responsein the area/percent of prostate damage from doses of 0.35 to 4.14 μg/gprostate; however, there was no significant increase from 4.14 to 25.79μg/g prostate (Table 15). The largest area of calculated damage wasobserved in monkeys receiving a dose of 4.14 μg/g prostate, in which asingle injection of 25 μL per lobe damaged approximately 50% of thetotal gland. The results suggest that the maximum damage may be limitedby the total distribution of the 25 μL injection volume per lobe ofprostate.

In treated areas where MPP5 induced significant infarction of normalglandular tissue, PSA staining was markedly decreased, while inadjacent, uninjured areas of the prostate, PSA staining was normal indistribution and degree. These results suggest that MPP5 killing ofcolumnar epithelial cells within the gland eliminates PSA production.The results also demonstrated that the distribution of MPP5 overlappedwith the infarct area at the mid- and high-dose levels as shown in FIG.38. At 15 days post-dose, no residual MPP5 was observed at the mid-doselevel. In addition, MPP5 did not appear to penetrate the prostatecapsule in any of the sections evaluated in this study.

TABLE 15 Area and Percent of Prostate Damage¹ from MPP5 Dose 0.35 μg/gprostate 4.14 μg/g prostate 25.79 μg/g prostate Area (cm²) Percent Area(cm²) Percent Area (cm²) Percent 0.33 20.2 0.78 46.6 0.57 63.4 0.36 30.20.87 47.9 0.32 23.2 0.20 13.8 0.89 54.9 0.51 41.0 0.46 36.8 1.23 56.60.47 51.0 Average 0.33 ± 0.11 25.3 ± 5.9 0.94 ± 0.20 51.5 ± 2.9 0.47 ±0.11 44.7 ± 9.8 (±Standard Deviation) ¹Damage (area/percent of totalgland) following injection of 25 μL per lobe

In treated areas where MPP5 induced significant infarction of normalglandular tissue, PSA staining was markedly decreased. In adjacentuninjured areas, PSA staining was normal in distribution and degree.These results suggest that MPP5 killing of columnar epithelial cellswithin the gland eliminates PSA production. However, MPP5 does not alterPSA production in uninjured areas, nor does it select for epithelialcells that produce lower levels of PSA. No PSA staining was observed inthe muscular cuff surrounding the urethra. This lack of PSA present inthe urethral tissue may partly explain the lack of any significantinjury to the urethra. Thus, a small volume (25 μL) injection of MPP5into a single lobe of the prostate can produce significant infarction ofa large area of PSA-producing glandular tissue in the normal monkeyprostate without significant injury to non-PSA producing structures(e.g., urethra).

Example 14 Toxicity of MPP5 in Dogs

A pilot toxicology study was performed in male beagle dogs in order toestablish the potential direct intraprostatic toxicity and MTD followinga single intraprostatic dose of MPP5. MPP5 was administered to malebeagle dogs (1/group) via intraprostatic injection (left lobe) of 0, 50,107, 200, or 400 μg in a dose volume of 100 μL (based on prostateweight, these doses were equivalent to 0, 22, 24.4, 40, and 72.2 μg/gprostate, respectively). The animals were observed for 1-week post-dose.There was no mortality, or treatment-related effects on clinicalobservations, body weight, food consumption, or clinical pathology.There was no apparent MPP5-related effect on prostate weight. Grosspathological changes were identified in the left lobe of the prostateand adjacent abdominal fat and consisted of dark areas of the prostateextending into the prostatic parenchyma. Adhesions to and/or dark areasin the abdominal fat were associated with the MPP5-related effects notedwithin the prostate. In the dog treated at 400 μg, the dark areas weremore numerous, and the left lobe of the prostate was enlarged. Increasesin the severity of these pathological changes were consideredattributable to the anticipated pharmacological effect of MPP5. Theredid not appear to be any significant extraprostatic toxicity. Overall,apparent treatment-related macroscopic changes were observed in theprostate, with limited associated effects on surrounding or adjacentabdominal fat tissue, with an increased severity at the 400 μg level.

The dog prostate shares structural similarities with the human prostate,including a 2-lobe structure, nature of the acinar ducts and theexistence of abundant stroma (Wientjes et al., 2005). Although manpossesses a higher fraction of stromal tissue than the dog prostategland, it is not known to what degree the architecture of fibrouspartitions, and the blood and lymphatic drainage patterns, differbetween man and dog prostate. Nonetheless, the dog has previously beendemonstrated as a useful model to study the effects of intraprostaticinjection (Rosser et al., 2004). However, in the case of MPP5, beagledogs did not appear overtly sensitive to the cytolytic effects of thiscompound. This is likely attributed to a lack of PSA expression in dogs(or other nonspecific enzymes capable of cleaving MPP5). The caninemodel shows that in the absence of PSA, MPP5 is not activated atextremely high doses, demonstrating the safety of the non-activatedprodrug. Rats and nonhuman primates, in contrast, are known to express aPSA-like kallikrein and PSA, respectively, and have been shown to besensitive to even low concentrations of MPP5. Thus, although the dogprostate is anatomically similar to the human prostate, it does notappear to exhibit a functional relation to the human gland in terms ofMPP5 activation, and, therefore, the dog was not pursued further as atoxicology model for MPP5. However, the dog study served to demonstratethat MPP5 appears to be pharmacologically inactive when not cleaved byPSA, providing confidence that MPP5 would not produce significanttoxicity if found in non-PSA-producing tissues.

Example 15 Toxicity of MPP5 in Monkeys

In order to establish the toxicity of MPP5 in an endogenousPSA-producing nonrodent species, an intraprostatic toxicity study wasconducted in male Cynomolgus monkeys (4/group) injected with 0.35, 4.14,or 25.79 μg MPP5/g prostate tissue as described in Example 7. Twoperineal injections were administered, one to each lobe of the prostate(25 μL/lobe). Preliminary results of this study are shown in Example 7.A description of the finalized results follows.

Following direct intraprostatic administration and a 2- or 14-dayobservation period, toxicity associated with MPP5 was confined to theprostate, with little damage to the surrounding tissues or other overtsystemic effects. Results of blood analysis indicated that maleCynomolgus monkeys express detectable levels of PSA and thatintraprostatic administration of MPP5 releases significant amounts ofPSA into the blood/serum. PSA levels returned to near baseline levels 10days following treatment. No treatment-related effects were observed atany dose level on clinical signs, body weight, ophthalmic condition,urinalysis, or electrocardiogram (ECG) evaluations. Serum chemistry andhematology assessments revealed a transient cellular and inflammatoryresponse. Transient acute phase immunological responses were observed inall groups on Study Day 3 and were attributed to inflammation associatedwith the surgical procedure and inflammation localized to the prostate.Increases in C-reactive proteins (CRP) noted on Study Day 3 weregenerally dose-related and consistent with the extent of mixed cellinflammation and necrosis of the prostate observed microscopically.

Gross and microscopic pathologic changes were observed in prostateglands at all MPP5 dose levels on Day 3. These changes werecharacterized by necrosis, hemorrhage, and mixed cell infiltrates andwere more severe in animals receiving MPP5 at the mid- and high doses.Monkeys in the low-dose group also exhibited histologic changes on Day 3that were consistent with repair, including regenerative hyperplasia andsquamous metaplasia in epithelial tissues and fibroplasia ininterstitial (mesenchymal) tissues. Repair in the mid- and high-dosegroups was minimal to absent on Day 3. By Day 15, necrosis had resolvedand repair was ongoing in the low-dose group. In the mid- and high-dosegroups, necrosis was ongoing on Day 15 and changes consistent withrepair were confined to the margins of necrotic lesions. In contrast,there were no changes in peri-prostatic or systemic tissues attributableto MPP5 on either Days 3 or 15.

Example 16 Immune Response to MPP5 in Monkeys

The potential immune response to MPP5 was also evaluated. The animalswere treated with MPP5 as described in Example 7. The potential immuneresponse to MPP5 was determined as follows.

Groups of monkeys received administration of various doses of MPP5directly into the prostate according to the table below. Serum(approximately 0.5 mL) was collected from the animals prior to the dayof injection and again at day 14 after injection. Serum was stored at−70° C. until assay. Immunoglobulin response was measured by ELISA asdescribed separately. Briefly, proaerolysin (0.5 μg/mL in phosphatebuffered saline) was bound to an EIA plate by coating overnight at 4° C.Non-specific binding was inhibited by coating with 5% BSA (Sigma) atroom temperature. A series of ten-fold dilutions (1:100-1:1,000,000) ofpooled normal monkey serum was used in quadruplicate to form acomparison curve for titer determination in serum from animals taken atvarious times after MPP5 administration. Samples of serum fromMPP5-treated monkeys were diluted ten-fold (1:100-1:1,000,000) toprovide concentrations with the normal serum. Peroxidase conjugated goatanti-rat IgG (Jackson ImmunoResearch) was bound to antibody that wasbound to the proaerolysin-coated well. Color was developed by additionof OPD peroxidase substrate according to manufacturer's instructions.Absorbance at 490 nm was measured on a Molecular Dynamics VERSAmaxmicroplate reader. Titer was defined as the dilution above which theabsorbance reading was less than that of normal pooled serum +2 standarddeviations for the same dilution.

Prior to MPP5 administration, all monkeys except one of thevehicle-control animals had no detectable titer. That control animalappears to have been exposed to an immunogen prior to the study as theappearance of a small titer was confirmed in the 14 day sample andreconfirmed by reassay. One of the two animals that received 1 μg/g MPP5exhibited a titer. Similarly, one of the two animals that received 5μg/g MPP5 exhibited a titer. Both animals that received 25 μg/g MPP5exhibited a titer.

Titers for each animal at the pre- and post-administration blood drawsare listed in Table 16.

TABLE 16 Antibody titers in monkeys after administration of MPP5 Dose(μg/g Route of Antibody Titer Group Monkey Prostate) Administration Day1 Day 14 1 6001  0 IP  1:1000*  1:1000* 1 8001  0 IP <1:100 <1:100 26003  1 IP <1:100  1:10,000 2 7002  1 IP <1:100 <1:100 3 6005  5 IP<1:100  1:10,000 3 8002  5 IP <1:100  1:100 4 6007 25 IP <1:100 1:10,000 4 8003 25 IP <1:100  1:10,000 *This animal demonstrated thesame small titer prior to administration.

FIG. 39 demonstrates the antibody titer in monkeys after administrationof MPP5. None of the titers were above 10⁴. This suggests thatintraprostatic administration of MPP5 does not elicit a strong immuneresponse. However, 4 of 6 animals treated exhibited a titer above pooledserum.

Antibody titers were noted in one of two monkeys treated with 0.35 or4.14 μg/g prostate and both animals that received 25.79 μg/g prostateexhibited a titer. Thus, administration of MPP5 induced a detectable,but low-titer immune response in some monkeys.

Based on the findings in Examples 11, 12 and 15, there was no indicationof extraprostatic toxicity at any dose level; thus, the systemic NOAELwas the highest MPP5 dose tested, 25.79 μg/g prostate (based on actualprostate weight). The effects observed in the prostate were bothdose-dependent and anticipated based on the known mechanism of action ofMPP5. Effects in the prostate were observed at all dose levels,including the lowest dose tested, 0.35 μg/g prostate, which damagedapproximately 25% of the prostate. Therefore, theLowest-Observed-Adverse-Effect Level (LOAEL) for local prostate effectswas 0.35 μg/g prostate in this study.

The previous examples describe the investigation of the drug metabolismand toxicokinetics of MPP5 following intravenous or intraprostaticadministration in male albino rats and following intraprostaticinjection in nonhuman primates. A summary of the nonclinical drugmetabolism and pharmacokinetics (DMPK) studies conducted with MPP5 ispresented in Table 17.

TABLE 17 List of nonclinical drug metabolism and pharmacokinetic studiesconducted with MPP5 Study Title Results An Acute Intraprostatic Providedtoxicokinetic data following a or Intravenous Bolus singleintraprostatic (2, 10, or 25 μg) Injection Toxicity or IV injection (25μg). MPP5 was not Study of MPP5 in the detectable followingintraprostatic Albino Rat (with a 1-, injection of 2 μg. C_(max)increased with 14-, or 28-Day increasing dose at 10 and 25 μg;Observation Period) however, the AUC at 10 μg was double [Example 6]that at 25 μg. After IV injection, t_(max) was 0 (immediate). TissueDistribution of Demonstrated that MPP5 had limited Radioactivity in Malesystemic bioavailability/distribution Sprague-Dawley Rats followingintraprostatic administration following Single and nonhomogeneousdistribution Injection of ¹²⁵I-MPP5 throughout the prostate. into theProstate Gland [Example 11] MPP5: A 2-Week Demonstrated a lack ofsystemic exposure Intraprostatic Acute following intraprostaticinjection (1, Toxicity Study in 5, or 25 μg/g prostate). All serumSexually Mature Male concentrations were below the LLOQ CynomolgusMonkeys (5.00 ng/mL), with the exception of 1 [Examples 7, 12, 13,low-dose animal which had concentrations and 15] above LLOQ at alltimepoints including pre-dose.

Example 17 Selection Of Dosage and Method of Administration of MPP5 inClinical Trials for BPH

An exemplary rationale for selecting a starting dose for clinical trialsof MPP5 in BPH is described below. An exemplary method of administeringMPP 5 is also described.

Based on the expression of PSA and the physiologic similarities betweenthe Cynomolgus monkey prostate and the human prostate, the single dosemonkey studies described herein are selected as the basis for estimatinga safe intraprostatic starting dose in humans. The dog was not sensitiveto the effects of MPP5 in comparison to rats and monkeys; thus, the dogwas not considered to be an appropriate model for estimating the safestarting dose of MPP5. Data from the rat studies described hereinsuggest that, despite the fact that the rat does not have the PSA codinggene, MPP5 is likely activated by a PSA-like S3 kallikrein identified inthe rat ventral prostate (Onozawa et al., 2001).

The starting dose of MPP5 in a BPH clinical trial is selected from therange of 0.03 μg/g to 0.25 μg/g prostate. A potential starting dose isset at 0.03 μg/g prostate, based on the application of a 10-fold safetyfactor to the lowest dose tested in the single dose monkey study (0.35μg/g prostate). In monkeys that received the 0.35 μg/g prostate dose, nosystemic toxicity was observed, while local prostate gland changes werenoted. While all 3 doses showed local ablation of prostate tissue, themid and higher doses demonstrated the most marked alterations and lackof healing at 14 days post injection. It was concluded that the lowestdose (0.35 μg/g prostate tissue) had the therapeutically usefulcombination of no systemic findings, either by histological orlaboratory analysis, and limited but clearly observable local prostaticeffect with approximately 25% ablation of the prostate. This wasconsidered a safe dose in the monkey. Using these data, a safety factorof at least 10-fold is applied and a starting dose of 0.03 μg of MPP5per gram of human prostate is chosen for the first cohort of the BPHtrial.

An exemplary method of administration of MPP5 in the BPH trials is thecommon transurethral route of administration with only 4 injections perdose (2 injections into each lateral lobe). For guidance duringinjection, for example, transrectal ultrasound can be used. The totalvolume to be administered in the BPH trial is 50 μL/gram of prostate. Toreduce backflow during injection, for example, a gel or viscousformulation can be used.

Although the invention has been described with reference to certainspecific embodiments, various modifications thereof will be apparent tothose skilled in the art without departing from the spirit and scope ofthe invention as outlined in the claims appended hereto.

The disclosure of all patents, publications, including published patentapplications, and database entries referenced in this specification arespecifically incorporated by reference in their entirety to the sameextent as if each such individual patent, publication, and databaseentry were specifically and individually indicated to be incorporated byreference.

1-46. (canceled)
 47. A method of decreasing prostate size in a subjectcomprising administering to said subject an effective amount of amodified pore-forming protein, said modified pore-forming proteinderived from a naturally-occurring pore-forming protein and comprisingone or more prostate-selective modifications selected from an activationsequence cleavable by a prostate-specific protease, and one or moreprostate-specific targeting domains capable of selectively targetingprostate cells, wherein said modified pore-forming protein is capable ofselectively killing prostate cells.
 48. A method of treating of benignprostatic hyperplasia (BPH) in a subject, comprising administering tosaid subject an effective amount of a modified pore-forming protein,said modified pore-forming protein derived from a naturally-occurringpore-forming protein and comprising one or more prostate-selectivemodifications selected from an activation sequence cleavable by aprostate-specific protease, and one or more prostate-specific targetingdomains capable of selectively targeting prostate cells, wherein saidmodified pore-forming protein is capable of selectively killing prostatecells.
 49. The method according to claim 48, wherein said modifiedpore-forming protein is administered in combination with one or moreother treatments for benign prostatic hyperplasia.
 50. The method ofclaim 47, wherein the naturally occurring pore-forming protein is aproaerolysin or an alpha toxin.
 51. The method of claim 47, wherein saidmodified pore-forming protein comprises an activation sequence cleavableby a prostate-specific protease.
 52. The method of claim 47, whereinsaid modified pore-forming protein comprises a prostate-specifictargeting domain.
 53. The method of claim 47, wherein said modifiedpore-forming protein comprises an activation sequence cleavable by aprostate-specific protease, and one or more prostate-specific targetingdomains.
 54. The method of claim 47, wherein said activation sequencecleavable by a prostate-specific protease functionally replaces a nativeactivation sequence of said naturally-occurring pore-forming protein.55. The method of claim 47 wherein said prostate-specific protease isprostate-specific antigen (PSA), prostate-specific membrane antigen(PSMA), or human glandular kallikrein 2 (hK2).
 56. The method of claim47, wherein said prostate-specific protease is prostate-specificantigen.
 57. The method of claim 47, wherein said prostate-specifictargeting domain is luteinizing hormone releasing hormone, or anantibody to prostate-specific membrane antigen.
 58. The method of claim47, wherein said modified pore-forming protein further comprises one ormore mutations in a binding domain of said naturally-occurringpore-forming protein.
 59. The method of claim 47, wherein said modifiedpore-forming protein further comprises an affinity tag.
 60. The methodof claim 47, wherein said modified pore-forming protein is formulatedfor intraprostatic administration.
 61. The method of claim 47, whereinsaid modified pore-forming protein is formulated for intravenousadministration.
 62. The method according to claim 47, wherein saidnaturally-occurring pore-forming protein is Aeromonas hydrophilaproaerolysin.
 63. The method according to claim 62, wherein saidmodified pore-forming protein comprises an activation sequence cleavableby a prostate-specific protease.
 64. The method according to claim 62,wherein said modified pore-forming protein further comprises one or moremutations in a binding domain of proaerolysin.
 65. The method accordingto claim 62, wherein said modified pore-forming protein comprises theamino acid sequence as set forth in SEQ ID NO:4.
 66. The methodaccording to claim 62, wherein said modified pore-forming proteincomprises an affinity tag.
 67. The method according to claim 62, whereinsaid modified pore-forming protein comprises the amino acid sequence asset forth in SEQ ID NO:31.
 68. The method according to claim 64, whereinsaid one or more mutations are selected from a mutation at positionY162, a mutation at position W324, a mutation at position R323, amutation at position R336, and a mutation at position W127.
 69. Themethod according to claim 64, wherein at least one mutation is R336A.70. A modified proaerolysin protein comprising one or more mutations ina large lobe binding domain, and one or more prostate-specificmodifications selected from a prostate-specific targeting domain capableof selectively targeting prostate cells and an activation sequencecleavable by a prostate-specific protease, wherein said modifiedproaerolysin is capable of selectively killing prostate cells.
 71. Themodified proaerolysin protein according to claim 70, wherein saidmodified proaerolysin protein is a modified Aeromonas hydrophilaproaerolysin.
 72. The modified proaerolysin protein according to claim71, wherein said large lobe binding domain is defined as amino acids 84to 426 of SEQ ID NO:2.
 73. The modified proaerolysin protein accordingto claim 72, wherein said one or more mutations in the large lobebinding domain are selected from a mutation at position Y162, a mutationat position W324, a mutation at position R323, a mutation at positionR336, and a mutation at position W127.
 74. The modified proaerolysinprotein of claim 70, wherein said modified proaerolysin proteincomprises a prostate-specific targeting domain.
 75. The modifiedproaerolysin protein of claim 70, wherein said modified proaerolysinprotein comprises an activation sequence cleavable by aprostate-specific protease.
 76. The modified proaerolysin protein ofclaim 70, wherein said modified proaerolysin protein comprises aprostate-specific targeting domain and an activation sequence cleavableby a prostate-specific protease.
 77. The modified proaerolysin proteinof claim 70, wherein said prostate-specific targeting domain isluteinizing hormone releasing hormone, or an antibody toprostate-specific membrane antigen.
 78. The modified proaerolysinprotein of claim 70, wherein said prostate-specific protease isprostate-specific antigen, prostate-specific membrane antigen, or humanglandular kallikrein 2.